ES2323588T3 - PROTEINS THAT BELONG TO THE BCL-2 FAMILY AND FRAGMENTS OF THE SAME, AND ITS USE IN PATIENTS WITH CANCER. - Google Patents

Abstract

Isolated immunogenically active peptide comprising a maximum of 15 amino acids, derived from the Bcl-2 protein, comprising a sequence selected from the group consisting of: WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI ( SEQ ID NO: 10) and NIALWMTEYL (SEQ ID NO: 11) for use as a medicine.

Description

Proteins that belong to the family Bcl-2 and fragments thereof, and their use in Cancer patients

Field of the Invention

The present invention generally relates to field of treatment and prophylaxis against cancer. In in particular, apoptosis regulatory proteins are provided isolated or peptide fragments thereof that can cause immune responses against cancer. Specifically, it provides the use of such proteins that belong to the family of Bcl-2 proteins and peptide fragments immunogenic thereof in diagnosis, prognosis and Cancer treatment

Previous technical and technical background

The development of cell resistance cancerous to a wide variety of chemotherapeutic agents It represents a major obstacle in satisfactory treatment of cancer Drug resistance is observed in a wide range of types of cancer cells. Many mechanisms contribute to the drug resistance, including drug inactivation, Extrusion of the drug by cell membrane pumps, mutations of pharmacological targets and failure to initiate apoptosis. The Apoptosis prevention can result from a variety of states, including membrane potential retention Mitochondrial and cytokine stimulation.

The search for proteins responsible for drug-resistant phenotypes has implicated the Bcl-2 antiapoptotic molecule. Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other tumors prone to apoptosis and, consequently, a poor prognosis in various cancers in humans. Bcl-2 belongs to a family of proteins, the Bcl-2 family, whose members regulate apoptosis (for review, see Reed et al (Oncogen, 1998, vol. 17, no. 24). The family includes both proapoptotic and antiapoptotic members WO89 / 58541 described the identification of an internal region of 63 amino acids in Bcl-XL involved in cytochrome C binding by Bcl-XL / Bcl-2 peptides It was shown that peptides thereof competed with the length peptides complete by cytochrome C binding. In US 5,856,171, the Bx-Bcl-2 binding Bax protein was used as a target for the development of apoptosis modulating agents, and peptides suitable for generation were disclosed of specific antibodies against Bax, WO 02/05835 described ion channels formed by members of the Bcl-2 family, involved in the release of cytochrome C and methods for identifying a compound that can interfere with these can ales WO 01/44282 described the DNA and protein sequences of BCL-G and the modulation of apoptosis by modulating the activity of BCL-G. US 5,470,955 described Mcl-1 and antibodies against Mcl-1 for use in diagnosis and treatment.

Although it is still difficult to reach a precise understanding of how Bcl-2 exercises its antiapoptotic effects, it has been found to be overexpressed in many cancers including lung, colorectal, cancers of prostate and breast as well as in leukemia and lymphomas.

Therefore, Bcl-2 is a factor critical cell, since the increased expression levels of that protein confers resistance to apoptotic stimuli, thereby contributing to the pathogenesis and progression of Cancer.

The process by which the system mammalian immune system recognizes and reacts against Foreign or foreign materials is complex. An important facet of system is the T cell response. This response requires that T cells recognize and interact with molecule complexes of the cell surface called leukocyte antigens humans (HLA) that constitute the major complex of Histocompatibility (CMH) human, and peptides. The peptides are they are derived from larger molecules, which are processed by cells, which in turn present the HLA / CMH molecule. The interaction of T cells and HLA / peptide complexes are restricted, requiring a T cell that is specific for a combination particular of an HLA molecule and a peptide. If not present a specific T cell, there is no T cell response even if its associated complex is present. Similarly not There is no answer if the specific complex is absent, but the T cell is present.

The mechanism by which T cells recognize Cellular abnormalities have also been implicated in cancer. By For example, in WO92 / 20356, a family of genes that are processed to give peptides that, in turn, are expressed on cell surfaces, and can lead to lysis of tumor cells by specific CTL. These genes are they call the MAGE family and they are said to code for "tumor rejection antigen precursors" or molecules "TRAP", and the peptides derived therefrom are called "tumor rejection antigens" or "TRA".

In Akatsuka Yoshiki et al (Journal of Exp. Med. 2003, vol. 197, No. 11) BCL2A1 derived peptides were identified as epitopes recognized by HLA-restricted CTL clones isolated from cancer patients who had received a bone marrow transplant . Likewise, an epitope derived from BCL2A1 has been described by Tetsuya et al (British journal of Hematology, 2004, vol. 124, No. 5) Sæterdal et al (PNAS. 2001, vol. 98. No. 23) described a TGF? RII peptide derived from a mutation with displacement of the reading frame that functions as a tumor-specific antigen in colorectal cancer.

In WO 94/05304, they are disclosed nonapeptides that bind to the HLA-A1 molecule. This reference discloses that, given the known specificity of particular peptides by particular HLA molecules, should Expect a particular peptide to bind to an HLA molecule, but not others. This is significant, since individuals Different have different HLA phenotypes. As a result, while identifying a particular peptide as capable of interacting with a specific HLA molecule has diagnostic and therapeutic repercussions, these are only relevant for individuals with that particular HLA phenotype.

WO 02/072627 presented a method for the identification of peptides for use in vaccines for induction of an anti-tumor cytotoxic effect. Be suggested peptides of several human proteins including members of the Bcl-2 family of proteins.

Therefore, it is well established that peptide epitopes derived from tumor associated antigens (TAAs) can be recognized as antigens by T lymphocytes cytotoxic (CTLs) in the context of CMH molecules. But nevertheless, although it is generally accepted that most if not all tumors they are antigenic, only some are in fact immunogenic in the sense that tumor progression is easily controlled by the immune system.

To overcome this limitation, they have started several immunotherapeutic studies, for example vaccinations with TAA derived peptides. For melanoma, the tumor for which it has characterized the largest number of TAAs defined by CTL, they have induced potent CTL responses against antigens by vaccination, and some patients experienced a remission Complete of his illness. However, most epitopes Peptides used in these vaccination trials are specific to melanocytes, and these peptides cannot be applied to tumors of non-melanocytic origin In addition, the expression of these TAAs is heterogeneous between tumors of different patients and may even vary between metastases obtained from a patient. But nevertheless, several antigens have been identified during the last two years tumor-specific peptides, which are expressed in several different cancers, that is, HER-2, Muc-1 and telomerase.

Apoptosis is a genetic suicide program cellular, and it has been suggested that apoptosis inhibition is a important mechanism involved in cancer formation prolonging the life of cells that favor the accumulation of transformative mutations The survivin is a member of the family of apoptosis inhibitor proteins (IAPs) recently identified. In a global gene expression analysis of approximately 4 million transcripts, the survivin as one of the main genes regulated by increase  invariably in many types of cancer but not in normal tissue. Solid malignant tumors that overexpress survivin include lung, colon, breast, pancreas and prostate cancer as well as hematopoietic malignant tumors. Additionally, it has reported that series of melanoma skin cancers and not Melanoma are invariably positive for survivin. The overexpression of survivin in most cancers in beings Human suggests a general role of apoptosis inhibition in tumor progression, a concept corroborated by observation that in the case of colorectal and bladder cancer, as well as neuroblastoma, the expression of survivin was associated with a unfavorable forecast. On the contrary, survivin cannot detected in normal adult tissues. These characteristics Qualify survivin as a suitable TAA for both Diagnostics as therapeutic.

Therefore, during the last decade it has identified a large number of TAAs that are recognized by CTLs in a way restricted by the major complex of histocompatibility (CMH). Since survivin is overexpressed in most cancers in humans and inhibition of their function results in an increase in apoptosis, this protein It can serve as a target for therapeutic CTL responses.

The survivin protein and the diagnostic use and potential therapeutic of it are disclosed in (1) and in the US 6,245,523. Survivin is a cytoplasmic protein 16.5 kDa containing a single BIR and a superhelix region highly charged terminal carboxyl of a RING finger, which inhibits the apoptosis induced by growth factor withdrawal (IL-3) when transferred in precursors of B cells. The gene encoding survivin is almost identical to the sequence of effector cell protease receptor 1 (EPR-1), but oriented in the opposite direction, what which therefore suggests the existence of two separate genes duplicates in a configuration head-to-head Consequently, the survivin can be described as a product of EPR-1 antisense. Functionally, the inhibition of the expression of survivin regulating by increase its transcript of EPR-1 natural antisense results in a massive apoptosis and decreased cell growth.

US 6,245,523 discloses the isolation of purified survivin and provides nucleic acid molecules encoding the survivin protein, and antibodies and other molecules that bind to survivin. US 6,245,523 also discloses anti-apoptotic active fragments of the survivin protein and variants thereof in which an N- or C-terminal amino acid residue has been inserted into, or within, the survivin sequence disclosed. It is specifically disclosed that such peptides must contain key functional residues required for apoptosis, ie Trp at position 67, Pro at position 73 and Cys at position 84. Schmidt et al (Blood. July 15, 2003, vol. 102. No. 2) further described that survivin and survivin peptides are recognized by specific cytotoxic T cells.

During the last decade, numerous trials clinicians have demonstrated the feasibility of specific vaccination of peptide to induce T cell responses anti-tumor in cancer patients. But nevertheless, the clinical evolution of the patients was not improved in the majority of the cases. This discrepancy has been explained in numerous cases. through immune escape mechanisms of tumor cells. For therapeutic strategies that select as antigens target that play an insignificant role in cancer growth, antigen-deficient cancer cell selection is a well recognized limitation.

       \ global \ parskip0.900000 \ baselineskip
    

However, in the case of cancer patients of breast, a paradoxical role of protein has been observed Bcl-2 In primary breast tumors it has been associated the negativity for Bcl-2 with a worse result clinical. Additionally, it has been reported that overexpression of the Bcl-2 protein is correlated with tumors  positive for estrogen receptors mediated by elements of estrogen receptor response in the gene promoter region of Bcl-2. The prognosis of positive tumors for estrogen is more favorable than that of negative tumors for estrogen Several possible explanations have been suggested for these seemingly paradoxical results, for example effects Bcl-2 inhibitors on cell proliferation, the regulation of the expression of Bcl-2 by estrogens and / or the presence of Bcl-2 antagonists that inhibit its cytoprotective function.

Still, previous studies have shown that overexpression of Bcl-2 in breast cancer is correlated with drug resistance, and that regulation by decrease of Bcl-2 by antisense oligonucleotides modulates drug sensitivity in association with apoptosis. . In addition, gene transfection of Bcl-2 in breast cancer cell lines has consistently resulted in enhanced apoptosis resistance. In addition, it has been described that the presence of another apoptosis inhibitor, the survivin protein in breast carcinoma was strongly associated with the expression of Bcl-2 and a reduced apoptotic index (AI) and poor overall survival. A similar association between survivin and Bcl-2 has been described in neuroblastoma, gastric cancer, colorectal cancer, and high-grade non-Hodgkin lymphoma. Therefore, in breast carcinoma as in most other cancers in humans, inhibition of apoptosis is a general feature, and the expression of anti-apoptotic genes, for example survivin and / or Bcl genes -2, may produce more pronounced antiapoptotic effects, as reflected in a reduced apoptotic index. Recently, survivin has been shown to be a target for spontaneous T-cell reactivity in patients with various cancers. These initial findings have been confirmed.
signed and secured later (by the present inventors and others) as in WO 2004/067023.

Summary of the invention

The present invention is based on the discovery that class I CMH-restricted peptides can be derived from a class of apoptosis regulatory proteins other than survivin, that is the Bcl-2 family of proteins, which can bind to a molecule of HLA of CMH class I and thereby causing immune responses of CTL in patients suffering from cancer diseases. These findings demonstrate that proteins belonging to the Bcl-2 family of proteins act as TRAP molecules, which are processed in vivo by cells to give peptides that have TRA functionality. These findings open the way to novel therapeutic and diagnostic approaches that can be applied in general in the control of cancer diseases.

The present invention discloses that Bcl-2 is a suitable target for immunotherapy against a range of cancer diseases. Bcl-2 It is a critical cellular factor and its expression is of importance for Tumor cell survival. So, Bcl-2 is an attractive target for vaccination because the immune escape through regulation by decrease or loss of expression of this protein would damage the sustained tumor growth. In addition, in studies that led to the present invention, the inventors sought and detected spontaneous reactivity of T cells in PBL against Bcl-2 derived peptides in cancer patients of breast using an ELISPOT test.

Accordingly, the present invention is refers in the first aspect to an immunogenically active peptide isolated comprising a maximum of 15 amino acids, derived from the Bcl-2 protein, which comprises a sequence selected from the group consisting of: WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10) and NIALWMTEYL (SEQ ID NO: 11) for use as a medicine in prevention or Cancer treatment

In a further aspect, the invention provides a pharmaceutical composition comprising the peptides of the invention.

It is also an aspect of the invention. providing a vaccine composition comprising a peptide immunogenically active isolated comprising a maximum of 15 amino acids, derived from the Bcl-2 protein, which It comprises a sequence selected from the group consisting of: WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10) and NIALWMTEYL (SEQ ID NO: 11) for use as a medicine in the prevention or treatment of cancer.

In still further aspects the invention relates to a diagnostic kit for the diagnosis ex vivo or in situ of the presence in a patient with T-cell cancer in PBL or in tumor tissue that are reactive with a member of the Bcl family of proteins. -2, the kit comprising the peptide of the invention as defined above; a complex of a peptide fragment of the invention and a class I HLA molecule or a fragment of such a molecule.

It is also an object of the invention provide a screening method in a patient with cancer of the presence of reactive T cells with a member of the family of Bcl-2 proteins, comprising the method put into contact a tumor tissue or a blood sample with a complex of the invention as defined above and detect the binding of the complex to tissue or blood cells.

       \ global \ parskip1.000000 \ baselineskip
    

In yet another aspect the invention provides the use of the protein or peptide fragment as defined in the present document in the manufacture of a medicine for Treatment of a cancer disease.

Detailed description of the invention

A main objective of the present invention is to provide isolated proteins that belong to the family of Bcl-2 proteins or a peptide fragment immunologically active thereof for use as a medicine in the prevention or treatment of cancer.

The Bcl-2 family of proteins It includes several proteins that regulate apoptosis. This family It includes both proapoptotic and antiapoptotic members. In the This report has studied the potential of this protein family as pharmaceutically active substances or from the point of view of diagnosis in cancer with particular reference to the Bcl-2 protein. In addition, it is described in detail the potential of Bcl-X_ {L} and Mcl-1 as a pharmaceutically active substance and from the point of view of diagnosis. However, it seems very there are likely to be immune responses similar to those observed against Bcl-2 protein or fragments of the same or that can be introduced in patients with cancer versus to other members of the Bcl-2 family of proteins, for example other antiapoptotic proteins such as Mcl-1 or Bcl-X_ {L}, which are also related to drug resistance and the overexpression in cancer. Consequently, the invention is refers to any member of the protein family Bcl-2, preferably any member antiapoptotic that can cause immune responses in cancer patients, for example a protein selected from group consisting of Bcl-2, Bcl-w, Mcl-1, Bfl-1 / A1, Bcl-b, Bcl2-L-10 and Bcl-X_ {L}, preferably selected from the group consisting of Bcl-2, Mcl-1, Bcl-w and Bcl-X_ {L}, more preferably selected from the group consisting of Bcl-2, Mcl-1 and Bcl-X_ {L}.

The antiapoptotic members of the Bcl-2 family exert their oncogenic effects by inhibiting apoptosis in cells that are normally destined to die, thereby promoting the accumulation of cells in vivo .

All members of the Bcl-2 family of proteins contain at least one of the four conserved motifs known as homology domains (BH) of Bcl-2 (BH1, BH2, BH3 and BH4). In addition to the presence of BH domains, preferred antiapoptotic molecules have an anchoring domain to the carboxyl terminal (TM) membrane. Antiapoptotic members such as Bcl-2 and Bcl-XL contain the four BH domains, together with the transmembrane domain. Multi-domain proapoptotic proteins such as Bax and Bak contain all domains except BH4. A second subset of proapoptotic proteins, known as "BH3 domain only" proteins (eg, Bad and Bid), consists of molecules that contain only the BH3 domain and lack other BH domains. Proapoptotic proteins such as Bcl-X_ {S} and Mcl-1S, which represent alternately cut and spliced forms of the bcl-x and mcl-1 genes, respectively, lack BH1 and BH2 domains. Additionally, Mcl-1S lacks a
transmembrane domain. Proteins belonging to the Bcl-2 family are described for example in reference 6.

Although it is preferred that the protein belongs to the Bcl-2 family of proteins have properties antiapoptotic, is also included within the present invention that the protein that belongs to the family Bcl-2 can be a proapoptotic protein, by example a protein selected from the group consisting of Bax, Bok / Mtd, Bad, Bik / Nbk, Bid, Hrk / DP5, Bim, Noxa, Bmf and PUMA / bbc3.

In a preferred embodiment of the invention, the protein that belongs to the protein family Bcl-2 is Bcl-2, preferably Human Bcl-2, more preferably Bcl-2 of the sequence with the registration number primary P10415 in the SwissProt database.

Since several cancers in humans express high levels of Bcl-2 and other members of the Bcl-2 family, immunotherapeutic strategies targeting these antigens can have wide applications clinics The main concern of such an approach it would be the induction of self-reactive immune responses. By therefore, the future of vaccination based on members of this family of proteins will depend on both therapeutic efficacy and type of side effects that could follow the immunization. When peptides derived from antigens for the first time were used melanocytic differentiation to treat patients with melanoma in stage IV it was anticipated that this could lead to destruction pronounced of melanocytes, which in turn would manifest itself clinically, for example as, vitiligo or retinitis. But nevertheless, clinical experience showed that the incidence of vitiligo in patients who received vaccinations was not significantly higher than the incidence of melanoma-associated hypopegmentation in patients who received other forms of treatment. Additionally, no serious side effects have been reported. in various vaccination trials against autoantigens.

In a useful embodiment, they are provided novel class I CMH restricted peptide fragments (in this document also called "peptides") which is characterized by having at least one of several characteristics, of which one is the ability to bind to the HLA molecule of class I to which it is restricted to an affinity as measured by the amount of the peptide that half of the maximum recovery of the HLA class I molecule (value C 50) which is a maximum of 50 µM as determined by the union-assembly test as described in this document. This assembly test is carried out as described above (2), and is based on the stabilization of the HLA molecule after loading the peptide into T2 cell line deficient in peptide transporter. Subsequently, stable HLA heavy chains are immunoprecipitated. folded correctly using antibodies dependent on the conformation and peptide binding is quantified.

This essay provides a simple means of examine candidate peptides to determine their ability to bind to an HLA allele molecule given to the affinity above. In preferred embodiments, the peptide fragment of the invention it is one that has a C_ {50} value, which is a maximum of 30 µM, such as a C_ {50} value, which is a maximum of 20 µM including C 50 values of at most 10 µM, at most 5 µM and at most 2 µM.

However, more preferred peptides according to the The present invention are peptides that can generate a response of specific T cells as determined by an assay ELISPOT, for example the ELISPOT test described later in the present document in example 1, section 4. Some peptides, although they do not join CMH with high affinity, they can result still to a T cell response as determined by ELISPOT Other peptides that can bind to CMH with high affinity also give rise to a T cell response as determined through ELISPOT. Both classes of peptides are preferred peptides according to the invention.

Therefore, preferred peptides according to the present invention are peptides that can generate a cell response Specific T as measured by an ELISPOT test, in which more than 50 specific peptide points are measured per 10 8 cells, more preferably for 10 7, even more preferably for 10 6, even more preferably for 10 5 cells, such as for 10 4 cells.

As mentioned earlier, the system of HLA represents the major histocompatibility system (CMH) human. Generally, CMH systems control a range of Features: transplant antigens, immune responses thymus-dependent, certain complement factors and predisposition to certain diseases. More specifically, the CMH encodes for three different types of molecules, that is molecules Class I, II and III, which determine the most characteristic CMH generals. Of these molecules, class I molecules are the so-called HLA-A molecules, HLA-B and HLA-C presented on the surface of most nucleated cells and thrombocytes.

The peptides of the present invention are characterized by their ability to join (being restricted a) an HLA molecule of the particular class I CMH. Therefore in one embodiment the peptide is one that is restricted to a HLA-A molecule of CMH class I including HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLA-Aw19, HLA-A23 (9), HLA-A24 (9), HLA-A25 (10), HLA-A26 (10), HLA-A28, HLA-A29 (w19), HLA-A30 (w19), HLA-A31 (w19), HLA-A32 (w19), HLA-Aw33 (w19), HLA-Aw34 (10), HLA-Aw36, HLA-Aw43, HLA-Aw66 (10), HLA-Aw68 (28), HLA-A69 (28). Throughout the bibliography simpler denominations are also used, using only the primary numerical designation, for example HLA-A19 or HLA-A24 instead of HLA-Aw19 and HLA-A24 (49), respectively. In specific embodiments, the peptide of the invention is restricted to a HLA species of CMH class I selected from group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.

The peptides of the invention can be derived for example from known sequences of a member of the family of Bcl-2 proteins (3). In a preferred embodiment of the invention, the peptide comprises (or more preferably consists of at) at most 200, preferably at most 100, more preferably at most 50, even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, such as at most 10, for example in the range of 9 to 10 contiguous amino acids of one of members of the Bcl-2 protein family mentioned above, preferably from Bcl-2 with the primary registration number P10415, Mcl-1 with the primary registration number Q07820 or Bcl-X_ {L} with the primary registration number Q07817 in the SwissProt database.

The selection of peptides that have potentially the ability to bind to an HLA molecule particular can be done by sequence alignment known to bind to a particular HLA molecule given to thereby reveal the predominance of some amino acids related in particular positions in the peptides. Such predominant amino acid residues are also referred to in the present document "anchor waste" or "reasons for anchor waste. "Following a relatively procedure simple of this type based on known sequence data that can be found in accessible databases, peptides they can be derived from protein family molecules Bcl-2, which is likely to bind to the molecule of HLA particular. Representative examples of such analyzes for determine a range of HLA molecules are given in table a continuation.

one

2

3

4

       \ vskip1.000000 \ baselineskip
    

Therefore, as an example, nonapeptides that have potentially the ability to join HLA-A1 they would have one of the following sequences: Xaa-T-D-Xaa-Xaa-Xaa-L-Xaa-Y, Xaa-T-E-Xaa-Xaa-Xaa-L-Xaa-Y; Xaa-S-D-Xaa-Xaa-Xaa-L-Xaa-Y or Xaa-S-E-Xaa-Xaa-Xaa-L-Xaa-Y (indicating Xaa any amino acid residue). In one way similarly, sequences can be designed that potentially have the ability to bind to any other HLA molecule.

It will be appreciated that the person skilled in the art will be able to identify additional "anchor waste grounds" for a given HLA molecule.

Therefore, in useful embodiments, the peptides of the invention include peptides, whose sequences comprise, for each of the specific HLA alleles listed in the table, any of the amino acid residues as indicated in the table.

Therefore, the peptides of the invention can be any of the above mentioned peptides that comprise contiguous sequences of family members of Bcl-2 proteins, having been exchanged in the range of 1 to 10, preferably in the range of 1 to 5, more preferably in the range of 1 to 3, even more preferably in the range of 1 to 2, even more preferably 1 amino acid by another amino acid, preferably in such a way that the peptide comprises one or more, preferably all anchor residues of a specific peptide of HLA-A given as indicated in the table previous.

       \ newpage
    

A non-limiting example of how to prepare peptides of members of the protein family Bcl-2 comprising anchor residues of a HLA-A specific peptide given is described in the Example 3 in the section "Response against peptides modified ". Therefore, in one embodiment of the invention, the peptide may be any peptide comprising a maximum of 200, preferably at most 100, more preferably at most 50,  even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, even more preferably at most 10 amino acids and comprising (or more preferably consisting of) a sequence selected from the group consisting of RLKRDWLVK (SEQ ID NO: 62), QSDEIISRY (SEQ ID NO: 63) and QSEEIISRY (SEQ ID NO: 64), more preferably selected of the group consisting of RLKRDWLVK (SEQ ID NO: 62).

Thus, a simple approach to identify peptides of the invention includes the following steps: select a particular HLA molecule, for example one that is produced at a high rate in a given population, carry out an alignment analysis as described. previously to identify the "reasons for anchor residues" in the Bcl-2 family of proteins, isolate or construct peptides of a suitable size that comprise one or more of the identified anchor residues and test the resulting peptides to determine (i) the ability to bind to the particular HLA molecule using the assembly assay as described herein, (ii) the ability of the peptides to elicit INF-γ producing cells in a PBL population of a cancer patient at a frequency of at least 1 per 10 4 PBL as determined by an ELISPOT assay as described herein. and / or (iii) the ability of the peptides to detect in situ in a CTL tumor tissue that are reactive with the epitope peptides being tested.

In specific embodiments, the peptide of the invention is a peptide derived from Bcl-2 restricted to HLA-A2 that has a sequence selected from the following: WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10), NIALWMTEYL (SEQ ID NO: 11).

In a preferred embodiment the peptide can be any peptide consisting of a maximum of 15, even more preferably at most 10 amino acids and comprising (or more preferably consisting of) a sequence selected from the group consisting of WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10), NIALWMTEYL (SEQ ID NO: 11), more preferably selected from the group consisting of NIALWMTEYL (SEQ ID NO: 11), YLNRHLHTWI (SEQ ID NO: 10), PLFDFSWLSL (SEQ ID NO: 8) and WLSLKTLLSL (SEQ ID NO: 6), even more preferably selected of the group consisting of PLFDFSWLSL (SEQ ID NO: 8) and WLSLKTLLSL (SEQ ID NO: 6).

The peptide can be any peptide that It consists of a maximum of 200, preferably a maximum of 100, plus preferably at most 50, even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, even more preferably as maximum 10 amino acids and comprising (or more preferably that consists of) a sequence selected from the group consisting of EMQVLVSRI (SEQ ID NO: 44), TAYQSFEQV (SEQ ID NO: 43), YLNDHLEPWI (SEQ ID NO: 42), RIAAWMATYL (SEQ ID NO: 45), WMATYLNDHL (SEQ ID NO: 46), VLVSRIAAWM (SEQ ID NO: 48) and VAFFSFGGAL (SEQ ID NO: 49), more preferably from the group consisting of TAYQSFEQV (SEQ ID NO: 43), YLNDHLEPWI (SEQ ID NO: 42), RIAAWMATYL (SEQ ID NO: 45), WMATYLNDHL (SEQ ID NO: 46), VLVSRIAAWM (SEQ ID NO: 48) and VAFFSFGGAL (SEQ ID NO: 49), even more preferably selected from the group that consists of TAYQSFEQV (SEQ ID NO: 43), VAFFSFGGAL (SEQ ID NO: 49), VLVSRIAAWM (SEQ ID NO: 48) and RIAAWMATYL (SEQ ID NO: 45) or selected from the group consisting of TAYQSFEQV (SEQ ID NO: 43) and WMATYLNDHL (SEQ ID NO: 46) or selected from the group consisting of YLNDHLEPWI (SEQ ID NO: 42).

The peptide can be any peptide that It consists of a maximum of 200, preferably a maximum of 100, plus preferably at most 50, even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, even more preferably as maximum 10 amino acids and comprising (or more preferably that consists of) a sequence selected from the group consisting of RIAAWMATY (SEQ ID NO: 50) and ALCVESVDK (SEQ ID NO: 51), more preferably selected from the group consisting of RIAAWMATY (SEQ ID NO: 50).

The peptide can be any peptide that It consists of a maximum of 200, preferably a maximum of 100, plus preferably at most 50, even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, even more preferably as maximum 10 amino acids and comprising (or more preferably that consists of) a sequence selected from the group consisting of YLREQATGAK (SEQ ID NO: 52), SITDVLVRTK (SEQ ID NO: 53), LISFGAFVAK (SEQ ID NO: 54), RLLFFAPTR (SEQ ID NO: 55), RTKRDWLVK (SEQ ID NO: 56) and DIKNEDDVK (SEQ ID NO: 57), more preferably selected from group consisting of RLLFFAPTR (SEQ ID NO: 55) and RTKRDWLVK (SEQ ID NO: 56).

The peptide can be any peptide that It consists of a maximum of 200, preferably a maximum of 100, plus preferably at most 50, even more preferably at most 25, even more preferably at most 20, even more preferably at most 15, even more preferably as maximum 10 amino acids and comprising (or more preferably that consists of) a sequence selected from the group consisting of PAEEEEDDLY (SEQ ID NO: 58), SPEEELDGY (SEQ ID NO: 59), QSLEIISRY (SEQ ID NO: 60) and AGVGAGLAY (SEQ ID NO: 61), more preferably selected from the group consisting of PAEEEEDDLY (SEQ ID NO: 58) and QSLEIISRY (SEQ ID NO: 60).

In additional useful embodiments, the peptide of the invention is a peptide, which is restricted to a molecule HLA-B of CMH class I including any of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44, HLA-B45, HLA-Bw46 and HLA-Bw47. In specific embodiments, the HLA-B species of Class I CMH to which the peptide of the invention can be attached is select from HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

In additional useful embodiments, the peptide of the invention is a peptide, which is restricted to a molecule HLA-C of the CMH class I including any of the following: HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLACw7 and HLA-Cw1.

Preferably, the peptide fragment of the invention comprises less than 50 amino acid residues, and more preferably comprises a maximum of 20 amino acid residues, such At most 10 amino acid residues. In realizations specific, the peptide is a heptapeptide, an octapeptide, a nonapeptide, a decapeptide or an undecapeptide.

The peptide of the invention is derived, such as mentioned above, from Bcl-2 or a fragment Of the same. The protein from which the peptide can be derived can be any Bcl-2 protein of any species animal in which the protein is expressed. In realizations preferred, the starting protein is from a mammalian species including a kind of rodent, rabbit and a kind of primate Just like human beings. Based on the protein sequence selected, the peptide of the invention is derived by any appropriate chemical or enzymatic treatment of the material starting protein that results in a peptide of a size suitable as indicated above, or can be synthesized by any peptide synthesis procedure conventional with which the expert is familiar.

The peptide of the invention may have a sequence that is a native sequence of the family member of Bcl-2 proteins from which it is derived. But nevertheless, peptides that have a greater affinity towards any molecule of Given HLA can be derived from such a native sequence modifying the sequence by substitution, deletion or addition of at least one amino acid residue, for example based on the procedure described above by which identify reasons for anchor residues with respect to the given HLA molecule.

A significant characteristic of the peptide of the invention is its ability to recognize or cause T cells of INF-γ producing response, ie cytotoxic T cells (CTL) that specifically recognize the particular peptide in a population of PBL or tumor cells of a cancer patient (target cells). This activity is determined easily subjecting a patient's PBL or tumor cells to a ELISPOT assay as described in reference (4) and in the following example. Before the test, it may be advantageous to stimulate the population of PBL or the tumor cells that are going to undergo assay by contacting the cells with the peptide that is going to to be tested Preferably, the peptide can cause or recognize INF-γ producing T cells a a frequency of at least 1 per 10 4 PBL as determined by an ELISPOT assay as used herein. More preferably the frequency is at least 5 times 10 4 PBL, most preferably at least 10 per 10 4 PBL, such as at least 50 or 100 per 10 4 PBL.

The ELISPOT trial represents a tool powerful for monitoring specific T cell responses of peptides derived from the Bcl-2 family. But nevertheless, although it has been shown that the reactivity of ELISPOT in most cases is correlated with the ability of CTLs to lyse target cells, the conclusive evidence for this concept may give only directly. Therefore, a main implication of  findings of the present document is that the peptides of the invention can be expressed and complexed with HLA molecules in cancer cells This makes these cancer cells sensitive to the destruction by CTL and highlights the potential utility of the immunization with proteins of the Bcl-2 family for Control the growth of neoplasms. The presence of responses of spontaneous CTL in PBL of breast cancer patients versus peptide epitopes derived from restricted Bcl-2 HLA corroborates the immunotherapeutic potential of these antigens tumor not only in patients with breast cancer, but also, already that members of the Bcl-2 protein family they are overexpressed in many cancers including lung cancers, colorectal, prostate and in leukemia and lymphomas, in a wide range of cancer diseases.

Consequently, in another preferred embodiment the peptide of the invention can cause cells producing INF-? In a PBL population of a patient who has a cancer disease in which a family is expressed of Bcl-2 proteins including a malignant tumor hematopoietic, for example, chronic lymphatic leukemia and leukemia chronic myeloid, melanoma, breast cancer, cervical cancer, cancer ovarian, lung cancer, colon cancer, pancreatic cancer and prostate cancer.

In addition to their ability to elicit immune responses in PBL populations, it is also contemplated that the peptides of the invention can elicit cytolytic immune responses in situ , that is, in solid tumor tissues. This can be demonstrated by providing HLA-peptide complexes, for example by multimerizing and providing a detectable label, and using such complexes for immunohistochemical stains to detect in a CTL tumor tissue that are reactive with the epitope peptide of the invention. Accordingly, an additional significant feature of the peptide of the invention is that it can perform in situ detection in a CTL tumor tissue that are reactive with the epitope peptide.

It is also contemplated that the peptides of the invention, in addition to its ability to bind to HLA molecules resulting in the presentation of HLA complexes and peptides on cell surfaces, complexes that in turn act as epitopes or targets for cytolytic T cells, may cause other types of immune responses, such as cell responses B resulting in the production of antibodies against complexes and / or a delayed type hypersensitivity reaction (DTH) The last type of immune response is defined as redness and palpable induration at the peptide injection site of the invention.

The vaccine composition according to the present invention may comprise a nucleic acid encoding a protein that belongs to the protein family Bcl-2 or a peptide fragment thereof. Saying nucleic acid can therefore code for any of the proteins and peptide fragments mentioned above. He nucleic acid can be for example DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is DNA or RNA.

The nucleic acids of the invention can be comprised within any suitable vector, such as a expression vector. Numerous vectors are available and The expert can select a vector useful for the specific purpose. The vector may be, for example, in the form of a plasmid, cosmid, viral particle or artificial chromosome. The sequence of appropriate nucleic acid can be inserted into the vector by a variety of procedures, for example, DNA can be inserted into a restriction endonuclease site (s) appropriate (s) using techniques well known in the art. Apart from the nucleic acid sequence according to the invention, the vector may further comprise one or more of a signal sequence, a origin of replication, one or more marker genes, an element enhancer, a promoter and a termination sequence of the transcription. The vector can also comprise sequences additional. The construction of suitable vectors containing one or more of these components uses ligation techniques conventional techniques known to the person skilled in the art. The vector is preferably an expression vector comprising the acid nucleic operably linked to a nucleic acid sequence regulator that directs its expression in a suitable cell. Within the scope of the present invention, said sequence of regulatory nucleic acid should be able to generally direct the expression in a mammalian cell, preferably a cell human, more preferably in a presenting cell of antigen.

In a preferred embodiment the vector is a viral vector. Said viral vector may comprise, in addition to the acid nucleic encoding a member of the protein family Bcl-2 or peptide fragment thereof, a second nucleic acid sequence encoding a polypeptide T cell stimulator The T cell stimulator polypeptide is preferably select from the group consisting of B7.1, ICAM-1 and LFA-3.

The vector may also be a bacterial vector, such as an attenuated bacterial vector. Attenuated bacterial vectors can be used in order to induce lasting immune responses of the mucosa at the sites of infection and persistence. Different recombinant bacteria can be used as vectors, for example, the bacterial vector can be selected from the group consisting of Salmonella , Lactococcus and Listeria . In general, the induction of immunity against the heterologous antigen VPH16 L1 or E7 could be demonstrated, with a strong induction of CTL and tumor remission in mice.

The invention also relates to a kit of parts that you understand

i)

any of the compositions of vaccine described herein and / or

ii)

any of the proteins that they belong to the Bcl-2 family of proteins described in this document and / or

iii)

any of the fragments peptides of the proteins of ii) described herein document and / or

iv)

any of the nucleic acids that encode for proteins of ii) or peptides of iii)

and an antineoplastic agent additional.

       \ vskip1.000000 \ baselineskip
    

The parts kit components are preferably comprised in individual compositions, without However, it is within the scope of this invention that all parts kit components are included within the same composition. Therefore, the Parts kit components can be administered so Simultaneous or sequential in any order.

The antineoplastic agent can be an agent used in chemotherapy or gene therapy, substances immunostimulants or antibodies. Immunostimulant substances they can be for example cytokines, such as selected cytokines  from the group consisting of GM-CSF, type I IFN, interleukin 12 and interleukin 15. The antibody is preferably an immunostimulant antibody such as antibodies anti-CD40 or anti-CTLA-4. The substance immunostimulatory can also be a substance that can deplete immune inhibition factors or cells (eg T cells regulators), said substance can be for example ubiquitin ligases E3. The ubiquitin-ligases E3 (HECT, RING and U-box) have emerged as molecular regulators key to immune cell function, and each one can be involved in the regulation of immune responses during infection by specific inhibitor molecules of selection such as target for proteolytic destruction. Several HECT proteins and RING E3 have now also been related to induction and maintenance of immune self-tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each one negatively regulates the proliferation and production of T cell growth factor

It is clear that the findings of the present invention provide the basis for therapeutic applications as well as diagnoses of the protein or the peptide fragment of the invention.

Consequently, in an additional aspect, the The present invention provides a pharmaceutical composition that comprises the protein or peptide fragment of the invention, in particular a pharmaceutical composition that, when administered to a cancer patient can cause an immune response against cancer disease including causing production in the patient vaccinated with effector T cells that have an effect cytotoxic against cancer cells.

As it is well known, that HLA molecules different are of different prevalence in the main populations of human beings, there is a requirement to identify peptide epitopes restricted to several class HLA molecules I to extend the cohort of patients that can be treated according to methods of the present invention. The characterization of multiple Bcl-2 epitopes with restriction elements of HLA different expands the clinical potential of this target antigen in two important ways: (i) increase the number of patients eligible for immunotherapy based on peptides derived from Bcl-2 The HLA-A2 antigen is expressed in approximately 50% of Caucasian populations and Asian, the two HLA-A1 antigens and HLA-A3 are expressed in approximately 25% of Caucasians and 5% of Asians, while the antigen HLA-A11 is expressed in approximately 15% of Caucasians and 30% of Asians. Although these figures cannot square due to coexpression, a combination of peptides restricted to a multiplicity of these would certainly encompass the majority of cancer patients, (ii) It is likely that the selection as collective target of several restriction elements of each patient reduce the risk of immune escape by loss of HLA alleles. The loss of a single HLA allele is a significant component of the CMH alterations described for cancer cells while total loss of expression of Class I is a fairly rare event. So, with the identification of epitopes of Bcl-2 restricted to different HLA alleles, it would be possible to select as target more than one HLA molecule simultaneously in patients with allele overlap.

Therefore, it would be possible to develop vaccines for multiple highly immunogenic epitopes. Preferably such vaccines should be designed to facilitate a simultaneous supply of the most suitable Bcl-2 derived peptides optionally in combination with other peptides and / or adjuvants suitable as described herein below document. The present invention encompasses such multiple vaccines. epitopes comprising peptides derived from Bcl-2 optionally in combination with other proteins or fragments of peptides that do not belong to or derived from the protein family Bcl-2 and / or adjuvants as described in continued in this document and / or epitopes restricted to CMH class II as described below.

There has been an increase in attention in the provocation of tumor-specific helper T cell immunity,  that is, vaccination with epitopes restricted to CMH class II a despite the fact that tumors generally do not express CMH from class II This is based on the recent finding that induction and the efficacy of the vaccine-induced antitumor response in many cases they require the cooperation of CD4 Th cells tumor-specific positives Therefore, an important factor which directs the development of vaccines that have one more composition complex is the desire to select multiple antigens as a target tumors for example designing vaccines that understand or that encode a set of epitopes of Th and CTL cells carefully selected.

Obviously, multiple epitope vaccines they constitute an effective way to generate immunity against epitopes derived from several different antigens without the need for introduce (genes that code for) proteins potentially dangerous such as oncoproteins. Such vaccines also allow a selective induction of immunity against T cell epitopes subdominant and cryptic, which can be especially important in the case of tumor-associated autoantigens for which it can there is tolerance for epitopes that occur in a manner prominent in normal tissues. In addition, the presenting cells of antigen may not present certain epitopes that are expressed in tumor cells due to functional differences between immunoproteasomes of antigen presenting cells and "constitutive" proteasomes present in most cells Tumor In the case of peptide-based vaccines, such epitopes can be administered in a "CMH-ready" way, which allows presentation through exogenous load independently  of the uptake and processing of antigens by cells host antigen presenters.

Since the peptides of the invention are relatively small molecules, it may be required in such compositions to combine the peptides with various materials such as adjuvants, to produce vaccines, immunogenic compositions, etc. Adjuvants, broadly defined, are substances that promote immune responses. Frequently, the adjuvant of choice is Freund's complete or incomplete adjuvant, or inactivated B. pertussis organisms, used for example in combination with alum precipitated antigens. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) on pages 61-63. Goding indicates, however, that when the antigen of interest is of low molecular weight, or is not immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include California limpet hemocyanin, bovine serum albumin, ovalbumin and poultry immunoglobulin. It has also been suggested that various saponin extracts are useful as adjuvants in immunogenic compositions. Recently, it has been proposed to use the granulocyte-macrophage colony stimulating factor (GM-CSF), a well-known cytokine, as an adjuvant (WO 97/28816).

The vaccine compositions according to the invention preferably comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are given below in the present document Therefore, the protein that belongs to the Bcl-2 family of proteins or peptide fragment of the same present in the composition can be associated with a carrier such as a protein or a cell presenting antigen such as for example a dendritic cell (DC) that can introduce yourself to the Bcl-2 family of proteins or peptide fragment thereof to a T cell.

Adjuvants are any substance whose mixture in the vaccine composition increases or otherwise modifies the immune response against the protein family Bcl-2 or peptide fragment thereof. The carriers are framework structures, for example a polypeptide or a polysaccharide, to which the family of proteins may be associated Bcl-2 or peptide fragment thereof.

The adjuvants could be selected, for example, from the group consisting of: AlK (SO4) 2, AlNa (SO4) 2, AlNH4 (SO4), silica, alum , Al (OH) 3, Ca 3 (PO 4) 2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11687, also called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '2'-dipalmitoyl-sn-glycer-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, also called MTP-PE), RIBI (MPL + TDM + CWS) in a 2% squalene / Tween-80.RTM emulsion. , lipopolysaccharides and their various derivatives, including lipid A, Freund's complete adjuvant (FCA), incomplete Freund's adjuvant, Merck's adjuvant 65, polynucleotides (e.g., poly IC and poly AU acids), Mycobacterium tuberculosis wax D, substances found in Corynebacterium parvum, Bordetella pertussis and members of the genus Brucella , liposomes or other emulsions d and lipids, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1 , interleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants for use with the invention include oil-based adjuvants / surfactants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants that include CpG olinucleotide sequences. Still other preferred adjuvants are adjuvants based on double stranded viral RNA, such as poly I: C. Imidazoquinilines are yet another example of preferred adjuvants. In addition, a preferred adjuvant are liposomes. The most preferred adjuvants are adjuvants suitable for use in humans.

Montanide adjuvants (all available from Seppic, Belgium), can be selected from the group consisting of Montanide ISA-51, Montanide ISA-50, Montanide ISA-70, Montanide ISA-206, Montanide ISA-25, Montanide ISA-720,  Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA 51 F, Montanide ISA 016D and Montanide IMS, preferably from the group consisting of Montanide ISA- 51, Montanide IMS and Montanide ISA-720, more preferably of the group consisting in Montanide ISA-51. Montanida ISA-51 (Seppic, Inc.) are adjuvants based on oil / surfactant in which different surfactants are combined or with a mineral oil that cannot be metabolized, an oil which can be metabolized, or with a mixture of both. Be prepare for use as an emulsion with an aqueous solution that comprises the protein that belongs to the protein family Bcl-2 or peptide fragment thereof. He surfactant is manide oleate. QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, MA) is a highly purified water soluble saponin that is treated as a aqueous solution The adjuvants QS-21 and Montanide ISA-51 can be provided in sterile vials of single use.

A general discussion of adjuvants is provides in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) on pages 61-63. Goding indicates, however, that when the antigen of interest is of low molecular weight, or is not immunogenic, the coupling to an immunogenic carrier. Examples of such carrier molecules include California limpet hemocyanin, bovine serum albumin, ovalbumin and poultry immunoglobulin. It has also been suggested that various saponin extracts are useful  as adjuvants in immunogenic compositions. Recently, it has proposed using the colony stimulating factor of granulocytes-macrophages (GM-CSF), a well-known cytokine, as an adjuvant (WO document 97/28816).

The desirable functionalities of adjuvants that can be used according to the present invention are listed in table a continuation.

TABLE 1 Modes of adjuvant action

5

6

A vaccine composition according to the present The invention may comprise more than one different adjuvant. Further, the invention encompasses a therapeutic composition comprising in addition any adjuvant substance including any of the previous or combinations thereof. It is also contemplated that the protein that belongs to the protein family Bcl-2 or peptide fragments thereof and the adjuvant can be administered separately in any sequence appropriate.

A carrier may be present regardless of an adjuvant. The function of a carrier can be for example increase the molecular weight of in particular peptide fragments in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity or to increase serum half-life. Further, a carrier can help present the protein that belongs to the Bcl-2 family or peptide fragments thereof to T cells. The carrier can be any suitable carrier known to one skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be, but not limited to, California limpet hemocyanin, serum proteins such as transferrin, serum albumin bovine, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins or hormones, such as insulin or palmitic acid. For the immunization of human beings, the carrier must be a physiologically acceptable carrier acceptable to humans and sure. However, tetanus toxoid and / or diphtheria toxoid they are suitable carriers in an embodiment of the invention. Alternatively, the carrier can be dextrans for example Sepharose

Consequently, the invention encompasses a therapeutic composition further comprising an adjuvant substance including any of the above or combinations of the same. It is also contemplated that the antigen, ie the peptide of the invention and the adjuvant can be administered simultaneously or separately in any appropriate sequence.

The choice of the antigen in the composition pharmaceutical of the invention will depend on parameters that may determined by the person skilled in the art. As it has been mentioned, each of the different peptides of the invention is presented on cell surfaces by an HLA molecule particular. As such, if a subject to be treated is typified With respect to the HLA phenotype, a peptide / peptides is selected that is known to bind to that HLA molecule particular.

Alternatively, the antigen of interest is select based on the prevalence of various HLA phenotypes in a given population. As an example, HLA-A2 is the most prevalent phenotype in the Caucasian population, and therefore, a composition containing a survivin-derived peptide that binds to HLA-A2 will be active in a large proportion of that population. However, the composition of the invention can also contain a combination of two or more peptides derived from survivin, interacting each specifically with a different HLA molecule to cover a larger proportion of the target population. Therefore, as examples, the composition pharmaceutical may contain a combination of a peptide restricted to an HLA-A molecule and a peptide restricted to an HLA-B molecule, for example including those HLA-A molecules and HLA-B corresponding to the prevalence of HLA phenotypes in the target population, such as for example HLA-A2 and HLA-B35. Further, the composition may comprise a peptide restricted to a HLA-C molecule.

It is contemplated that immunogenic compositions useful of the invention, in addition to a peptide derived from members of the Bcl-2 family of proteins as defined in this document may comprise an amount immunologically effective member of the protein family Bcl-2 as such, as defined herein document or an immunogenic fragment thereof.

The amount of the immunogenic peptide of the invention in the pharmaceutical composition may vary, depending of the particular application. However, a single dose of immunogen is preferably any from about 10 \ mug to about 5000 \ mug, more preferably from about 50 \ mug to about 2500 \ mug, such as from about 100 \ mug to about 1000 \ mug. Modes of administration include intradermal administration, subcutaneous and intravenous, implantation in the form of a formulation of liberation in time, etc. Any and all forms of administration known in the art are encompassed in the present document They are also encompassed any and all conventional pharmaceutical forms known in the art that are suitable for the formulation of a peptide composition Injectable immunogenic, such as solutions and forms lyophilized, suspensions or emulsion forms containing, if required, carriers, diluents, preservatives, adjuvants, pharmaceutically acceptable buffer components, etc.

Pharmaceutical compositions can be prepared and administered using any conventional protocol known to a person skilled in the art. In example 5 it is given an example of non-limiting preparation of a composition of vaccine according to the invention, as well as an example of administration not limiting it as a vaccine. One skilled in the art you will appreciate that the protocol can be easily adapted to anyone of the vaccine compositions described herein document.

In a further embodiment of the invention, the Pharmaceutical composition of the invention is useful for treating a cancer patient, in which, during the evolution of cancer in that patient, cancer cells have developed a reduced susceptibility to a cancer drug chemotherapy active and / or radiotherapy.

The pharmaceutical composition of the invention it can advantageously comprise at least one protein additional immunogenic or peptide fragment thereof selected from a protein or peptide fragment that does not belong a or derived from the Bcl-2 family of proteins, including a protein involved in the regulation of apoptosis cell or a peptide fragment derived therefrom. As a For example, an additional protein or peptide of this type is survivin as defined above, or a fragment peptide thereof. In specific embodiments, a peptide Additional immunogenic survivin derivative is a peptide restricted to HLA-A2 that has a sequence selected from the following: FLKLDRERA (survivina_ {101-109}) (SEQ ID NO: 12), TLPPAWQPFL (survivina_ {5-14}) (SEQ ID NO: 13), ELTLGEFLKL (survivina_ {95-104}) (SEQ ID NO: 14), LLLGEFLKL (SEQ ID NO: 15) and LMLGEFLKL (SEQ ID NO: 16). (The denominations in brackets indicate the positions of the residues in the survivin protein as disclosed in US document 6,245,523). LLLGEFLKL (SEQ ID NO: 15) is a sequence derived from survivina_ {96-104} replacing "T" in the peptide position 2 by "L" and LMLGEFLKL (SEQ ID NO: 16) is derives from survivin_ {96-104} replacing "T" in position 2 for "M". In specific embodiments additional, the additional immunogenic survivin derived peptide it is a survivin-derived peptide restricted to HLA-B35 that has a selected sequence of following: CPTENEPDL (survivina_ {46-54}) (SEQ ID NO: 17), EPDLAQCFF (survivina_ {51-59}) (SEQ ID NO: 18), CPTENEPDY (SEQ ID NO: 19) and EPDLAQCFY (SEQ ID NO: 20). (The denominations in brackets indicate the positions of the residues in the survivin protein as disclosed in the US 6,245,523). CPTENEPDY (SEQ ID NO: 19) is a sequence survivin derivative_ {46-54} substituting "L" at the C-terminal end of the peptide by "Y" and EPDLAQCFY (SEQ ID NO: 20) is derived from survivina_ {51-59} replacing a residue "F" at the C-terminal 2 end by "Y".

Even in further embodiments, the peptide additional is an HLA-A1 restricted peptide that It has a sequence selected from the following: survivina_ {38-46} (Sur38Y9) (C changes to Y in P9, MAEAGFIHY) (SEQ ID NO: 21), survivina_ {47-56} (Sur47Y10) (Q changes to Y in P10, PTENEPDLAY (SEQ ID NO: 22)), survivina_ {92-101} (Sur92-101) (QFEELTLGEF) (SEQ ID NO: 23) and survivin_ {93-101} (Sur93T2 (E changes to T in P2, FTELTLGEF (SEQ ID NO: 24)). The peptide of the invention may also be a peptide restricted to HLA-A3 such as survivin_ {18-24} (Sur18K10) (F changes to K at P10, RISTFKNWPK (SEQ ID NO: 25) and / or a HLA-A11 restricted peptide such as survivina_ {53-62} (Sur53-62) (DLAQCFFCFK) (SEQ ID NO: 26) and / or a peptide restricted to HLA-A2 such as survivin_ {18-28} (South18-28) (RISTFKNWPFL) (SEQ ID NO: 27).

However, in a preferred embodiment of the invention, the vaccine compositions do not comprise survivin or fragments of it.

Other useful additional peptides include the known apoptosis inhibitor polypeptide ML-IAP that has a fairly selective expression, and is detected in melanomas. Therefore, fragments of ML-IAP that can cause a cell response Specific T, that is a cytotoxic T cell response or a Auxiliary T cell response can optionally be included in The composition of the present invention. Peptide fragments Useful ML-IAPs include any of the ML-IAP fragments described in the application for WO2004 / 089980, preferably ML-IAP_ {245} (RLQEERTCKV) (SEQ ID NO: 28), ML-IAP_ 280 (QLCPICRAPV) (SEQ ID NO: 29), ML-IAP_ {90} (RLASFYDWPL) (SEQ ID NO: 30), ML-IAP_ {154} (LLRSKGRDFV) (SEQ ID NO: 31), ML-IAP_ {230} (VLEPPGARDV) (SEQ ID NO: 32), ML-IAP_ {98} (PLTAEVPPEL) (SEQ ID NO: 33), ML-IAP_ {34} (SLGSPVLGL) (SEQ ID NO: 34), MLIAP_ {54} (QILGQLRPL) (SEQ ID NO: 35), ML-IAP_ {99} (LTAEVPPEL) (SEQ ID NO: 36), ML-IAP_ {83} (GMGSEELRL) (SEQ ID NO: 37) and ML-IAP_ {200} (ELPTPRREV) (SEQ ID NO: 38).

Other useful additional peptides include TRAG-3 and peptide fragments thereof. TRAG-3 exists in at least two ways alternatively cut and spliced and the peptides of all TRAG-3 splicing shapes are useful as additional peptides In particular, fragments of any form of splicing of TRAG-3, in which said fragments can elicit a specific T cell response, it is say a cytotoxic T cell response or a response from Auxiliary T cells can optionally be included in the Composition of the present invention.

Additionally, the composition according to the present invention can be provided as a multiple epitope vaccine comprising epitope restricted to class I and epitopes restricted to class II as defined above in the present document

The immunoprotective effect of the composition of the invention can be determined using several approaches, for example as described in WO 97/28816, cited previously. A satisfactory immune response can also determined by the appearance of DTH reactions after immunization and / or detection of antibodies that recognize specifically the peptide (s) of the composition of vaccine.

In preferred embodiments, the pharmaceutical composition of the invention is a vaccine composition. Therefore, the pharmaceutical composition may be a vaccine or immunogenic composition that can elicit an immune response against a cancer disease. As used herein; The term "vaccine or immunogenic composition" refers to a composition that causes at least one type of immune response directed against cancer cells. Therefore, an immune response of this type can be any of the types mentioned above: a CTL response generating CTL that can recognize the HLA / peptide complex presented on cell surfaces resulting in cell lysis, ie the vaccine causes the production in the vaccinated subject of effector T cells that have a cytotoxic effect against cancer cells; a response from
B cell leading to the production of antibodies against cancer; and / or a type of immune response DTH.

In useful embodiments, a immunogenic response directed against cancer disease administering the peptide of the invention or loading molecules of CMH class I in antigen presenting cells (APC) of patient, isolating PBL from the patient and incubating the cells with the peptide before injecting the cells back into the patient, or well isolating APC precursors of the patient and differentiating the Professional APC cells using cytokines and the antigen before inject the cells back into the patient.

Therefore, it is an aspect of the invention provide vaccine compositions comprising cells antigen presenters comprising a protein that belongs to the Bcl-2 family or a peptide fragment of the same or a nucleic acid encoding said protein or said peptide fragment. The antigen presenting cell can be any cell that can present an antigen to a T cell. Preferred antigen presenting cells are cells dendritic Dendritic cells (DC) can be prepared and used in a therapeutic procedure according to any suitable protocol, for example as described hereinafter document. The skilled artisan will appreciate that the protocol can adapt for use in patients with different type of HLA and different diseases

Dendritic cells (DC) are pulsed with HLA restricted 50 µg / ml peptide (synthesized according to GMP quality) for 1 h at 37 ° C peptide and 5 x 10 6 cells were administered subcutaneously on day 1 and 14, subsequently every 4 weeks, additional leukophoresis after 5 vaccinations. The DC generation for clinical use and quality control can realizers essentially as described in the reference 5.

Thus, in one embodiment of the present invention, a method of treating cancer patients is one in which the peptide is administered by presenting the peptide to the antigen presenting cells (APC) of the ex vivo patient followed by injecting the treated APCs. in this way again in the patient. There are at least two alternative ways of doing it. An alternative is to isolate APC from the cancer patient and incubate (load) the class I CMH molecules with the peptide. Loading the class I CMH molecules means incubating the APCs with the peptide so that the APCs with the class I specific CMH molecules for the peptide will bind to the peptide and therefore can present it to the T cells. Subsequently, the APCs they are injected back into the patient. Another alternative mode is based on recent discoveries made in the field of dendritic cell biology. In this case, monocytes (which are precursors of dendritic cells) are isolated from the patient and differentiated in vitro into professional APCs (or dendritic cells) by the use of cytokines and the antigen. Subsequently, the DC generated in vitro with the peptide is pulsed and injected into the patient.

Due to the fact that family members of Bcl-2 proteins appear to be expressed in a range of Forms of cancer, it is very likely that the vaccines of the invention can be provided to control any type of disease of cancer in which such proteins are expressed. Therefore, as examples, the vaccine composition of the invention is immunologically active against a hematopoietic malignant tumor including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer and cancer prostate.

From the above description, the expert will easily understand that the proteins and / or peptides of the invention are useful as cancer diagnostic tools. Therefore, the peptides of the invention provide the basis for developing diagnostic and prognostic procedures widely applicable with respect to cancer diseases. Therefore, in other useful embodiments, the composition of the invention is a composition for the diagnosis ex vivo or in situ of the presence in a cancer patient, for example based on the detection of reactive T cells with members of the protein family. Bcl-2 family between PBL or in tumor tissue.

Consequently, a diagnostic kit for the ex vivo or in situ diagnosis of the presence in a patient with T-cell cancer reactive with members of the Bcl-2 family among PBL or in tumor tissue is provided, in additional aspects. it comprises one or more peptides of the invention, and a detection method in a cancer patient of the presence of such reactive T cells, the method comprising contacting a tumor tissue or a blood sample with a complex of a peptide of the invention and a class I HLA molecule or a fragment of such a molecule and detect the binding of the complex to the tissue or blood cells.

Another useful diagnostic or prognostic approach is based on the generation of antibodies in a heterologous animal species, for example murine antibodies directed against a peptide derived from members of the human Bcl-2 protein family of the invention, which can then be used, for example to diagnose the presence of cancer cells that present the peptide. For such immunization purposes, the amount of peptide may be less than that used in the course of in vivo treatment, as mentioned above. In general, a preferred dose may range from about 1 µg to about 750 µg of peptide. It is also possible to produce monoclonal antibodies based on immunization with a peptide of the invention. Accordingly, the present invention also relates to a molecule, in particular a monoclonal or polyclonal antibody including a fragment thereof, which can specifically bind a peptide of the invention and a molecule that can block such a binding, for example an antibody generated against the monoclonal or polyclonal antibody directed against a peptide of the invention. The invention further relates to isolated T cell receptors that can specifically bind to a peptide or protein of the invention as well as isolated nucleic acids encoding them. Such T cell receptors can be cloned for example from specific protein or peptide T cells using conventional techniques well known to the expert.

In one aspect, the invention also relates to isolated T cells comprising T cell receptors that can specifically bind to any of the proteins belonging to the Bcl-2 family and / or peptide fragments thereof described herein. . The isolated T cells are preferably T cells that have expanded in vitro . Methods for expanding T cells in vitro are well known to the expert. Such T cells may be useful in particular in the treatment of cancer by adaptive transfer or autologous cell transfer. Therefore, the invention also relates to treatment methods comprising administering T cells comprising T cell receptors that can specifically bind to a protein belonging to the Bcl-2 family or peptide fragments thereof to an individual, such As a human being who is suffering from cancer. The invention further relates to the use of T cells comprising T cell receptors that can specifically bind to a protein belonging to the Bcl-2 family or peptide fragments thereof for the preparation of a medicament for the treatment of cancer. Autologous cell transfer can be performed essentially as described in reference 7.

In one aspect, the invention provides a complex of a peptide of the invention and an HLA molecule of class I or a fragment of such a molecule, which is useful as a reagent of diagnosis as described above. A complex of this type can be monomeric or multimeric.

The present invention provides the means to relieve or cure a cancer disease. Consequently, a Additional aspect of the invention is to provide a method for treat a cancer disease associated with the expression of a member of the Bcl-2 family of proteins, including as examples: a hematopoietic malignant tumor including leukemia chronic lymphatic and chronic myeloid leukemia, melanoma, cancer breast, cervical cancer, ovarian cancer, lung cancer, cancer of colon, pancreatic cancer and prostate cancer, comprising the method administer to a patient suffering from the disease a effective amount of the pharmaceutical composition according to the invention, a molecule that can specifically bind a peptide of the invention and / or a molecule that can block the binding of a molecule of this type.

In some cases it will be appropriate to combine the treatment method of the invention with a treatment conventional cancer such as chemotherapy, radiotherapy, treatment with immunostimulant substances, gene therapy, antibody treatment and treatment using cells dendritic Since the high expression of members of the Bcl-2 family of proteins in tumor cells is correlated with drug resistance, the combination of a Bcl-2-based immunotherapy as given to know by the present invention with cytotoxic chemotherapy It could be an effective approach to treat cancer.

In one aspect, the invention relates to methods. to monitor immunization, said method comprising the stages of

i)

provide a blood sample of An individual

ii)

provide a protein that belongs to the Bcl-2 family of proteins or a peptide fragment thereof, said protein or said being peptide any of the proteins or peptides described in the present document

iiii)

determine whether said sample of blood comprises antibodies or T cells that comprise receptors T cell that specifically bind to the protein or the peptide

iiv)

determine that way if generated an immune response to said protein or said peptide in that individual.

The individual is preferably a human being, for example a human being who has been immunized with a protein that belongs to the Bcl-2 family of proteins or a peptide fragment thereof or a nucleic acid encoding for said protein or said peptide.

The invention will now be illustrated by the following non-limiting examples and the drawings in which

Figure 1 shows the identification of BLA-2 HLA-A2 binding peptides. The heavy chain bands of CMH class I were quantified using a Phosphorimager device. The amount of heavy chain of HLA-A2 stabilized is directly related with the binding affinity of the added peptide. Peptide binding positive control restricted to HLA-A2 HIV Pol 476 (square in black) was compared with peptides Bcl_ {172} (triangle in black), Bcl_ {180} (circle in black) and Bcl_ {200} (blank circle) and

Figure 2 illustrates the T cell response against peptides Bcl 172, Bcl 180, Bcl 208 and Bcl_ {214}. PBL of 15 patients with breast cancer were analyzed. T lymphocytes were stimulated once with the peptide before plating at 10 5 cells per well in triplicate or either without or with peptide. The average number of points peptide specific (after subtracting points without added peptide) was calculated for each patient using the InmunoSpot® series analyzer 2.0 (CTL Analyzers, LLC, Cleveland, USA).

Figure 3 illustrates the T cell response versus Bcl-2 as measured by ELISPOT of INF- γ. PBL of ten patients were analyzed with HLA-A2 positive LLC, three patients with AML positive for HLA-A2 and two cancer patients pancreatic (PC). Peptides Bcl 208 (A) and Bcl_ {214} (B). T lymphocytes were stimulated once with the peptide before plating at 10 5 cells per well in triplicate or without or with peptide. The average number of specific peptide points (after subtracting points without peptide added) was calculated for each patient using the analyzer InmunoSpot® 2.0 series (CTL Analyzers, LLC, Cleveland, USA). The patients responding to treatment (defined as the number average of specific points of antigen ± 1/2 of the deviation standard> 25 by 10 5 lymphocytes) are marked as squares in black while unresponsive individuals They are marked as blank squares.

Figure 4 illustrates the detection of CTL specific for Bcl-2 by ELISPOT of granzyme B. T lymphocytes were stimulated from four patients with breast cancer in a different late stage (b19, b20, b22, b16) and a healthy control (h1) once with the peptide before plating at 10 5 cells per well in triplicate either without or with peptide Bcl_ {208} (A) or Bcl_ {214} (B). The average number of points of peptide specific granzyme B (after subtracting points without added peptide) was calculated for each patient using the analyzer InmunoSpot® 2.0 series (CTL Analyzers, LLC, Cleveland, USA). Patients responding to treatment (defined as the number average of specific points of antigen ± 1/2 of the deviation standard> 25 by 10 5 lymphocytes) are marked as squares in black while unresponsive individuals They are marked as blank squares.

Figure 5 illustrates the cytolytic capacity of CTL specific to Bcl-2.bcl_ {208}. They were isolated CTL PBL reagents of a breast cancer patient using beads magnetic coated with HLA-A2 / bcl_208. A) It analyzed the isolated mass culture to determine lysis T2 cell specific with (square in black) or without (square in white) bcl 208 peptide. B) Lysis by isolated T cells by bcl_208 of the breast cancer cell line positive for HLA-A2 MDA-MB-231 (circle in black) and the negative breast cancer cell line for HLA-A2 ZR75-1 (circle in White).

Figure 6 illustrates the T cell responses restricted by HLA-A2 versus Bcl-X_ {L} as measured by ELISPOT of INF- γ. PBL of twelve individuals were analyzed healthy, eighteen breast cancer patients (BC patients), six melanoma patients and two patients with pancreatic cancer (PC patients). All individuals were positive for HLA-A2 Peptides were examined Bcl-X_ {L173-182} (YLNDHLEPWI) (SEQ ID NO: 48) (A), Bcl-X_ {L141-150} (VAFFSFGGAL) (SEQ ID NO: 49) (B), Bcl-X_ {L161-170} (VLVSRIAAWM) (SEQ ID NO: 48) (C), and Bcl-X_ {L165-174} (RIAAWMATYL) (SEQ ID NO: 45) (D). T lymphocytes were stimulated a peptide time before plating at 10 5 cells per well in triplicate or without or with the relevant peptide. The average number of specific peptide points was calculated (after subtract points without added peptide) for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA.). Patients who respond to treatment (defined as the average number of specific points of antigen ± 1/2 of the standard deviation> 25 by 10 5 lymphocytes) are marked as squares in black, while individuals who do not Respond are marked as blank squares.

Figure 7 illustrates the detection of CTL specific to Bcl-X_ {L} through ELISPOT of Granzyme B. T lymphocytes were stimulated from three patients with different breast cancer (BC35, BC36 and BC17) once with the peptide before plating at 3x105 cells per well per triplicate either without or with peptide Bcl-X_ {L173-182} (YLNDHLEPWI). Be calculated the average number of specific granzyme B points of peptide (after subtracting points without added peptide) for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA). Patients who respond to treatment (defined as the average number of specific antigen points ± 1/2 of the standard deviation> 25 per 10 5 lymphocytes) they are marked as squares in black, while individuals that do not respond are marked as blank squares.

Figure 8 illustrates the analysis of CD8 positive cells, specific for Bcl-XL, in PBL of a breast cancer patient. PBL from patient BC36 was stimulated once with Bcl-X L173-182 in vitro and CD8 + cells were isolated before analysis. FACS staining of the culture using an anti-CD8 antibody and the HLA-A2 / Bcl-X1717-182 pentamer complex revealed that 95.5% of the cells were CD8 positive and 0.24% of them. They were positive for the pentamer (A). The HLA-A2 / HIV pentamer was used as a negative control (B). The cell culture was further analyzed by means of ELISPOT (C).

Figure 9 illustrates the T cell responses restricted by HLA-A2 versus Bcl-X_ {L} as measured by ELISPOT of INF- γ. PBL of twelve individuals were analyzed healthy, eighteen breast cancer patients (BC patients), six melanoma patients and two patients with pancreatic cancer (PC patients). All individuals were positive for HLA-A2 Peptides were examined Bcl-X_ {L118-126} (TAYQSFEQV) (SEQ ID NO: 43) (A) and Bcl-X_ {L169-178} (WMATYLNDHL) (SEQ ID NO: 46) (B). T lymphocytes were stimulated a peptide time before plating at 10 5 cells per well in triplicate or without or with the relevant peptide. The average number of specific peptide points was calculated (after subtract points without added peptide) for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA.). Patients who respond to treatment (defined as the average number of specific points of antigen ± 1/2 of the standard deviation> 25 by 10 5 lymphocytes) are marked as squares in black, while individuals who do not Respond are marked as blank squares.

Figure 10 illustrates the T cell responses restricted by HLA-A3 versus Bcl-X_ {L} as measured by ELISPOT of INF- γ. T lymphocytes were stimulated a peptide time before plating at 10 5 cells per well in triplicate either without or with the peptide Bcl-X_ {L165-173} (RIAAWMATY) (SEQ ID NO: 50). PBL of seven healthy individuals, five were examined breast cancer patients, four melanoma patients, two patients with pancreatic cancer, and five patients with myeloma multiple. All individuals were positive for HLA-A3. The average number of points was calculated peptide specific (after subtracting points without added peptide) for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA).

Figure 11 illustrates the T cell responses restricted by HLA-A3 versus Mcl-1 as measured by ELISPOT of INF- γ. T lymphocytes were stimulated a peptide time before plating at 3x105 cells per well in triplicate or without or with the peptide. Be examined PBL of ten healthy individuals, six cancer patients of breast (BC), two patients with pancreatic cancer (PC), and six patients with CLL against the peptide Mcl-195-103 peptide (left) and the Mcl-1300-308 peptide (right). All individuals were positive for HLA-A3. Be calculated the average number of specific peptide points (after subtract points without added peptide) for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA.). Patients who respond to treatment (defined as the average number of specific points of antigen ± 1/2 of the standard deviation> 25 by 10 5 lymphocytes) are marked as squares in black, while individuals who do not Respond are marked as blank squares.

Figure 12 illustrates the T cell responses restricted by HLA-A1 versus Mcl-1 as measured by ELISPOT of INF- γ. T lymphocytes were stimulated a peptide time before plating at 3x105 cells per well in triplicate either without or with the peptide Mcl-1166-175 or Mcl-1177-185. PBL were examined for six healthy individuals, four patients with breast cancer (BC) and seven patients with melanoma against the peptide Mcl-195-103 (left) and the peptide Mcl-1300-308 (right). All the Individuals were positive for HLA-A1. The number average peptide specific points (after subtracting points no added peptide) was calculated for each patient using the InmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA.). Patients who respond to treatment (defined as the average number of specific points of antigen ± 1/2 of the standard deviation> 25 by 10 5 lymphocytes) are marked as squares in black, while individuals who do not Respond are marked as blank squares.

       \ vskip1.000000 \ baselineskip
    

Examples Example 1 Immune responses against Bcl-2 in breast cancer patients Materials and methods 1. Patients

Peripheral blood lymphocytes were collected (PBL) of patients with breast cancer. PBLs were isolated using separation with Lymphoprep, were typed for HLA (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients He received immunotherapy before taking the blood sample.

       \ vskip1.000000 \ baselineskip
    

2. Assembly test for peptide binding to CMH class I molecules

Peptide binding affinity was measured Synthetics (Invitrogen, Carlsbad, CA, USA) to molecules HLA-A2, metabolically marked with [35 S] -methionine, in the assembly test, as described above. The essay is based on the peptide-mediated stabilization of empty HLA molecules released after cell lysis, from the T2 cell line deficient in TAP. Folded HLA molecules were immunoprecipitated stably using the W6 / 32 AcM dependent on the conformation, specific to HLA class I, and separated by gel electrophoresis and isoelectric focusing (IEF). They were quantified CMH heavy chain bands using the ImageGauge program Phosphorimager (FUJI photo film Co., Carrollton, TX, USA). The band intensity is directly related to the amount of class I CMH complex bound to recovered peptide during the rehearsal Subsequently, the degree of stabilization of HLA-A2 is directly related to the affinity of peptide binding added. The recovery of HLA-A2 in the presence of 50, 5, 0.5, 0.05 µM of relevant peptide The C 50 value for each peptide was calculated as the concentration of peptide sufficient to obtain half of The maximum stabilization.

       \ vskip1.000000 \ baselineskip
    

3. PBL antigenic stimulation

To extend the sensitivity of the ELISPOT assay, PBLs were stimulated once in vitro before analysis. On day 0, PBL or crushed lymph nodes were thawed and plated in 2 ml / well at a concentration of 2 10 6 cells in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Maryland), 5% heat inactivated human serum and 2 mM L-glutamine in the presence of 10 µM peptide. Two days later, 20 IU / ml recombinant interleukin-2 (IL-2) was added to the cultures (Chiron, Ratingen, Germany). Cultured cells were tested for their reactivity in ELISPOT on day 12.

       \ vskip1.000000 \ baselineskip
    

4. ELISPOT test

The ELISPOT assay was used to quantify the effector cells that release interferon-? peptide epitope-specific as described previously (4). In summary, plates were coated 96-wells with nitrocellulose bottom (MultiScreen MAIP N45, Millipore, Hedehusene, Denmark) with antibody anti-IFN-? (1-D1K, Mabtech, Nacka, Sweden). They washed wells, were blocked by X-vivo medium and were added cells in duplicate at different concentrations cell phones. Peptides were then added to each well and they incubated the plates overnight. The next day, it was discarded the medium and the wells were washed before the addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed and added avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies) to each well. The plates were incubated at RT for 1 hour and the NBT / BCIP enzyme substrate (Gibco, Life Technologies) at each well and incubated at RT for 5-10 min. Put on end the reaction by washing with running water after the emergence of dark purple dots Points were counted using the ImmunoSpot® 2.0 series analyzer (CTL Analyzers, LLC, Cleveland, USA) and the frequency of specific CTLs of peptide from the numbers of cells that formed points. All assays were performed in triplicate for each antigen. peptide

       \ vskip1.000000 \ baselineskip
    

5. Results Bcl-2 derived peptide binding to HLA-A2

The amino acid sequence of the Bcl-2 protein to determine epitopes HLA-A2 Nona and Decamer Peptides plus likely, using the specific anchor residues of HLA-A2 main (2). Thirteen were synthesized peptides derived from Bcl-2 and examined for determine its binding to HLA-A2 by comparison with the high affinity positive control epitope for HLA-A2, HIV-1 pol_476-484 (ILKEPVHGV) (SEQ ID NO: 39) by The assembly test. The assembly test is based on the stabilization of the class I molecule after loading different peptide concentrations in the T2 cell line deficient in TAP Subsequently, CMH heavy chains are immunoprecipitated stable folded correctly using antibodies dependent on the conformation The degree of stabilization of CMH molecules of class I is directly related to the binding affinity of Peptide added as shown as an example in Figure 1. The peptide concentration required to achieve half of the maximum recovery of class I CMH molecules (C50 value) was 0.7 µM for HIV-1 pol_ {476-484} (table 1). Eight derived peptides of Bcl-2 joined with a high affinity almost similar to positive control; Bcl_ {224}, Bcl_ {85}, Bcl_ {222}, Bcl_ {218}, Bcl_ {220}, Bcl_ {214}, Bcl_ {124} and Bcl_ {172} (C 50 = 0.7, 1, 1, 2, 1, 3, 1 and 2 µM, respectively) (Table 1). Peptides Bcl 80, Bcl 208 and Bcl 180 are joined only with an intermediate or weak affinity (C 50 = 36, 7 and 20 µM, respectively). Two of the peptides examined (Bcl_ {216}, Bcl_ {200}) did not join HLA-A2 absolutely. A list of the peptides is shown in Table 1 included in this study:

       \ vskip1.000000 \ baselineskip
    

TABLE 1 Peptides examined in this study

7

a range of values listed in subscripts indicates the position of the first amino acid in the sequence

^ b The value C 50 is the concentration of the peptide required to achieve the half of the maximum connection to HLA-A2

       \ vskip1.000000 \ baselineskip
    

CTL responses to peptides derived from BCL-2 in breast cancer patients treated with chemotherapy

Using the secretion test of IFN-? ELISPOT, the presence of specific T cell responses to derived peptides of Bcl-2 in peripheral blood T cells of breast cancer patients This method has been previously highly effective when tumor-specific CTLs were identified in cancer patients.

PBL of 15 HLA-A2 positive breast cancer patients were stimulated once in vitro before the ELISPOT test. This procedure was chosen to extend the sensitivity of ELISPOT as described (4). Since many CTL epitopes described are in fact low affinity peptides, the thirteen peptides deduced from Bcl-2 were included in the first line of experiments. Responses were detected against Bcl_172, Bcl_ {180}, Bcl_ {208} and Bcl_ {214} and only data on these peptides are given in Figure 2. Spontaneous CTL responses were detected against Bcl_172 in PBL of eight of the patients (50%), and against Bcl_ {180} in four of the patients (approx25%) (Figure 2). However, the most frequent responses were detected against Bcl_ {208} and Bcl_ {214}, since twelve (approx80%) of the patients harbored a detectable CTL response against Bcl_ {208} and eleven of the patients ( approx75%) harbored a response against Bcl_ {214} (figure 2).

       \ vskip1.000000 \ baselineskip
    

Example 2 Bcl-2 immunogenicity in patients with Cancer Summary

In this example, the reactivity of spontaneous T cells against Bcl-2 in blood peripheral of patients suffering from non-tumor types related, that is, pancreatic cancer, AML and CLL. Additionally, it is shown that these T cells reactive against Bcl-2 are in fact cytotoxic effector cells, peptide specific. Therefore, Bcl-2 can serve as an important and widely applicable target for immunotherapeutic strategies against cancer, for example in combination with conventional radio and chemotherapy.

       \ vskip1.000000 \ baselineskip
    

Introduction

The Bcl-2 family comprises several key actors in the regulation of apoptosis and includes both proapoptotic and antiapoptotic molecules. Bcl-2 is a critical cellular factor that contributes to the pathogenesis and progression of cancer. In the present study, it examined the natural cellular immunogenicity of Bcl-2 in cancer patients.

       \ vskip1.000000 \ baselineskip
    

Methods Patients

PBL were isolated using separation with Lymphoprep, were typed for HLA (Department of Immunology Clinic, University Hospital, Copenhagen, Denmark) and se Frozen in FCS with 10% DMSO. None of the patients received immunotherapy before taking the blood sample. The Informed consent of patients before any of these measures. Peripheral blood lymphocytes (PBL) were collected of thirteen breast cancer patients positive for HLA-A2 presenting progressive disease with distant metastases that defined stage IV disease; the Most patients had more than one tumor location (8/13 patients). The previous treatment included chemotherapy, endocrine treatment and radiotherapy. Eight patients were treated previously with chemotherapy, while five patients had received only endocrine treatment and no chemotherapy before his Inclusion in the study. In addition, twelve patients were included positive for HLA-A2 with operable breast cancer located and blood samples were collected before the Primary surgery and chemotherapy. Additionally, it collected PBL from two patients with pancreatic cancer positive for HLA-A2 presenting progressive disease with distant metastases that defined the disease in stage IV. Finally, PBL was collected from ten patients with fresh CLL diagnosed HLA-A2 and three with AML before treatment. As controls, they served PBL from twelve healthy individuals positive for HLA-A2.

       \ vskip1.000000 \ baselineskip
    

ELISPOT of granzima B

The ELISPOT test of granzyme B (GrB) was used to measure the cytotoxicity of antigen specific CTL such as It is described. In summary, 96-well plates were coated with nitrocellulose bottom (MultiScreen MAIP N45, Millipore) with GrB capture antibody (BD Biosciences, Brondby, Denmark). The wells were washed and blocked by means X-vivo with 5% human serum. The added cells at different cell concentrations. Then T2 cells and peptides were added to each well and the plates for 4 hours, the medium was discarded and the wells before the addition of the GrB detection antibody (BD Biosciences). The plates were incubated for 2 hours, washed and Avidin-horseradish peroxidase (BD) was added Biosciences) to each well. The plates were incubated at RT for 1 hour, AEC substrate reagent (BD Biosciences) was added to each well and incubated at RT for 5-10 min. Put on end the reaction by washing with running water after the emergence of Red dots. The points were counted and the frequency of CTL peptide specific as for the ELISPOT of IFN- γ. All trials were performed by duplicated or triplicate for each peptide antigen.

       \ vskip1.000000 \ baselineskip
    

Isolation of peptide specific T cells

Antigen specific cells were isolated by medium of magnetic beads coated with Bcl_208 / HLA-A2 as described previously. Biotinylated monomers (Prolmmune, Oxford, RU) to magnetic beads coated with streptatividin (Dynabeads M-280, Dynal A / S, Oslo, Norway) incubating 2.5 µg of monomers with 5x106 beads in 40 µl of PBS, for 20 min. at room temperature. They were washed the magnetic complexes three times in PBS in a magnetic field (Dynal A / S, Oslo, Norway) and subsequently mixed with PBL, to a ratio of 1:10 in PBS with 5% BSA, and they were rotated very gently for 1 h. They washed very gently three times antigen specific CD8 + T cells that were associated with magnetic complexes The isolated cells were resuspended numerous times in X-vivo with 5% HS and it incubated for 2 h, before the magnetic beads were released and remove them from cell suspensions. Cells were cultured isolated in a 48-well plate in X-vivo, HS 5% and 10 6 antigen presenting cells based on artificial cells coated with antibody anti-CD28, anti-CD3 antibody (K32 / 41 BBL) expressing the 4-1BB ligand (4-1 BBL) (provided courtesy of Dr. Carl H. June, Department of Pathology and Laboratory Medicine, University of Pennsylvania). One day after isolation, it was added IL-2 20 units / ml, and on day 5 underwent test the ability of these cells to destroy target cells any in conventional 51 Cr release assays.

       \ vskip1.000000 \ baselineskip
    

Cloning by dilution limit

CTL clones were established from the cultures isolated by limit dilution in 96-well plates using irradiated PBMC as feeder cells in the presence of IL-2 40 IU / ml and PHA 1 / cup / ml in X-vivo with 5% HS. New medium was added and IL-2 to clones every 3-4 days.

       \ vskip1.000000 \ baselineskip
    

Cytotoxicity test

Release tests of [51] Conventional to determine mediated cytotoxicity by CTL as described elsewhere in this document. The target cells were T2 cells with or without the relevant peptide, the positive breast cancer cell line for HLA-A2 MDA-MB-231 and the negative breast cancer cell line for HLA-A2 ZR75-1. Both lines of Breast cancer expressed Bcl-2 as examined by reverse transcription PCR (data not shown).

       \ vskip1.000000 \ baselineskip
    

Results CTL responses to peptides derived from Bcl-2

To examine whether specific T cells from Bcl-2 were also present in PBL of patients with leukemia, PBL of ten patients with positive CLL were examined for HLA-A2 and three patients with AML to determine  the reactivity against the two peptides bcl 208 and bcl 214. Responses to Bcl-2 were present in five of the patients with CLL and two of the patients with AML (figure 3). In addition, PBL from two pancreatic cancers were examined. and it was identified that both patients harbored a CTL response against the bcl 208 and bcl 214 peptides (Figure 3). From similarly, PBL from twelve healthy individuals were examined positive for HLA-A2. Surprisingly, it was detected a weak CTL response against the bcl 208 peptide in one of healthy individuals (data not shown).

       \ vskip1.000000 \ baselineskip
    

Release of granzyme B specific to Bcl-2 in PBL

Using GrB ELISPOT, it was assessed whether bcl-2 specific T cells detected in PBL They showed cytotoxic function. Therefore, PBL of three were analyzed of breast cancer patients reactive with bcl-2 (patients no .: 19, 20 and 22) to determine reactivity against the two epitopes bcl_208 and bcl_ {214} (figure 4). In all three patients, responses could be detected against both peptides with a frequency at approximately 50-140 CTL peptide specific for 10 5 PBL. As a control, a patient was included (patient no .: 16), in which only one response could be detected against bcl_ {172} but no against bcl_ {208} and bcl_ {214} in the ELISPOT of IFN-? And a healthy control (h1). As it expected, no GrB release was detected against bcl_208 or bcl_ {214} neither in the patient with breast cancer # 16 nor in the sound control

       \ vskip1.000000 \ baselineskip
    

The functional capacity of CTL reagents with Bcl-2

To further characterize the functional capacity of CTL reactive with Bcl-2, these cells were enriched by means of magnetic beads coated with HLA-A2 / bcl 208 complexes as described. Cells were stimulated once with peptide in vitro before isolation. A small fraction of the isolated cells was cloned by limit dilution. Expanding cultures were examined for recognition of T2 cells either without peptide or pulsed with bcl 208 in a GrB ELISPOT. Several of these clones showed a specific recognition of T2 cells pulsed with bcl_208 (data not shown). However, unfortunately these clones could not be expanded for further analysis.

One day after isolation, IL-2 was added to the remaining cells, and on day 5 the ability of the cells to destroy peptide-loaded T2 cells was tested in conventional 51 Cr release assays. For this purpose, either unloaded T2 cells or T2 cells loaded with bcl 208 peptide served as targets. This assay revealed that only pulsed T2 cells were destroyed with bcl 208 (Figure 5a). These in vitro- stimulated and bcl 208-reactive T cells were additionally used to test the ability to destroy the MDA-MB-231 breast cancer cell line, which expresses Bcl-2 and is positive for HLA-A2 . Enriched T cells effectively lysed MDA-MB-231 cells, while instead no cytotoxicity was observed against the ZR75-1 breast cancer cell line, which is negative for HLA-A2 and expresses Bcl-2 (Figure 5b ).

       \ vskip1.000000 \ baselineskip
    

Example 3 Immunogenicity of Bcl-X (L) in patients with cancer Summary

In this example, it is shown that Bcl-X_ {L} is a target for the recognition of T cells in cancer patients. Therefore, they are described spontaneous cytotoxic T cell responses restricted by HLA-A2 and HLA-A3 against epitopes peptides derived from Bcl-XL by means of flow cytometry stains and ELISPOT. Therefore, the cellular immune responses against inhibitors of apoptosis as the proteins of the Bcl-2 family seem to represent a general phenomenon in cancer, and in Consequently, this group of proteins represents target proteins universal attractive for cancer immunotherapy. Additionally, since the elevated expression of these proteins in cells is correlated with drug resistance, the combination of immunotherapy with cytotoxic chemotherapy is a way Very attractive to treat cancer.

       \ vskip1.000000 \ baselineskip
    

Introduction

Bcl-X L antiapoptotic protein is produced from the long alternative splicing form of the bcl-x gene, while proaptotic Bcl-X S is derived from the alternative short splicing form thereof. gen. Bcl-X_ {L} plays an important role as it has been directly related to resistance to conventional forms of treatments and poor prognoses. Functional inhibition of Bcl-X L restores the apoptotic process and makes neoplastic cells sensitive to chemo and radiotherapy, while manipulating cancer cell lines to express high levels of Bcl-X L results in multi-drug resistance phenotype. Increased expression of Bcl-X L has been reported in a variety of different malignant tumors including AML and multiple myeloma as well as solid cancers such as bladder cancer, breast cancer, pancreatic cancer and melanoma.

Ideal targets for immunotherapy are products gene silenced in normal tissues, overexpressed in cells cancerous, and directly involved in the progression and tumor cell survival.

       \ vskip1.000000 \ baselineskip
    

Materials and methods Patients

Peripheral blood lymphocytes were collected (PBL) of patients suffering from cancer of different origin and of healthy controls and were isolated using separation with Lymphoprep, were typified for HLA (Department of Clinical Immunology, Hospital University, Copenhagen, Denmark) and froze in FCS with 10% DMSO. None of the patients received immunotherapy before the taking of the blood sample. Consent was obtained informed of patients before any of these measures.

       \ vskip1.000000 \ baselineskip
    

Flow Cytometry (FACS)

PBL from a breast cancer patient with the relevant peptide was stimulated once in vitro and on day seven CD8 + cells were isolated from PBL using the Dynal negative CD8 isolation kit (Dynal Biotech ASA, Oslo, Norway). The resulting CD8 positive T cell culture was stained with PE-coupled Pro5? CMH pentamers (ProImmune, Oxford, RU), followed by antibody staining with the fluorochrome-coupled MCAs: CD8-APC and CD3-FITC ( Becton Dickinson, Immunocytometry Systems, San José, CA). Both stains were performed in PBS + 2% FCS, for 30 min., 4 ° C, in the dark. The CMH Pro5? Pentamer complexes were used: HLA-A2 / Bcl-X_ {L173-182} (YLNDHLEPWI) (SEQ ID NO: 42) and HLA-A2 / HIV-1 pol 476-484} (ILKEPVHGV) (SEQ ID NO: 39). Samples were analyzed on BD FACS aria, using DIVA software (BD, San José, CA).

       \ vskip1.000000 \ baselineskip
    

Results Responses of spontaneous CTL against peptides derived from Bcl-X_ {L}

The bcl-x gene is transcribed into two mRNAs through alternative splicing. Bcl-X L antiapoptotic protein is produced from the long alternative splicing form, while proaptotic Bcl-X S is derived from the short alternative splicing form of this gene. The largest BCL-X L protein product differs from the Bcl-X S protein in an inserted region (amino acids 126-188). Therefore, to investigate whether Bcl-XL is a natural target for T cells in cancer patients, this inserted region (including nine amino acids at each end) was inspected to determine suspected HLA-A2 epitopes using residues of specific HLA-A2 specific anchorage. Subsequently, seven peptides deduced from Bcl-X_L (Bcl-X_ {L158-166} (EMQVLVSRI) (SEQ ID NO: 44), Bcl-X_ {L118-126} (TAYQSFEQV) (SEQ ID NO: 43), Bcl-X_ {L173-182} (YLNDHLEPWI) (SEQ ID NO: 42), Bcl-X_ {L165-174} (RIAAWMATYL) (SEQ ID NO: 45), Bcl-X_ {L169-178} ( WMATYLNDHL) (SEQ ID NO: 46), Bcl-X_ {L161-170} (VLVSRIAAWM) (SEQ ID NO: 48), Bcl-X_ {L141-150} (VAFFSFGGAL) (SEQ ID NO: 49)) and se they inspected PBL of patients with HLA-A2 + cancer of different origin by means of ELISPOT against these peptides. Previously, this method has been shown to be highly effective in identifying tumor-specific CTLs in cancer patients. In fact, strong and frequent CTL responses were detected against four of the peptides examined (Bcl-X_ {L173-182}, Bcl-X_ {L141-150}, Bcl-X_ {L161-170} and Bcl-X_ { L165-174}) in patients with cancer of different origin (Figure 6). In total, fifteen of the eighteen patients with HLA-A2 + breast cancer harbored an immune response against at least one of these four Bcl-X L peptides (patients responding to treatment are defined as the average number of cells antigen-specific ± 1/2 of the standard deviation> 25 per 10 5 cells). Likewise, four of the six melanoma patients examined and one of the two pancreatic cancer patients examined harbored an immune response against at least one of these four peptides. Therefore, nine of the eighteen breast cancer patients examined harbored an immune response against Bcl-X_ {L173-182}, while two of the six patients with HLA-A2 + melanoma examined harbored an immune response against this peptide ( figure 6a). Four of the eighteen breast cancer patients examined harbored an immune response against Bcl-X1414-150, while PBL responses were detected from one of the two pancreatic cancer patients examined. No response could be detected in PBL from any of the five melanoma patients examined against this peptide (Figure 6b). Likewise, a response was detected in PBL from six patients with breast cancer and one patient with pancreatic cancer examined against Bcl-X L161-170 (Figure 6c). Finally, four patients with breast cancer, two patients with melanoma and one patient with pancreatic cancer hosted a response to Bcl-X_ {L165-174} (Figure 6d). As a control, PBL from 12 healthy HLA-A2 + individuals were examined. Importantly, no responses were detected against any Bcl-X_ {L173-182}, Bcl-X_ {L141-150}, Bcl-X_ {L161-170} or Bcl-X_ {L165-174} peptides healthy individuals (figure 6)

       \ vskip1.000000 \ baselineskip
    

Specific granzyme B release from Bcl-X_ {L} in PBL

Using GrB ELISPOT, it was assessed whether Bcl-XL specific T cells detected in PBL showed cytotoxic function. Therefore, PBL of two of the breast cancer patients reactive against Bcl-X_ {L (patients no .: 35 and 36) to determine reactivity against Bcl-X_ {L173-182} (figure 7). In Both patients were able to detect responses against Bcl-X_ {L173-182} with a frequency at approximately 50-100 specific CTL of peptide by 3x105 cells. As a control, a patient (patient no .: 17), in which only one response versus Bcl-X_ {L141-150} but not in front of Bcl-X_ {L173-182} in the ELISPOT of IFN- γ. As expected, it was not detected GrB release versus Bcl-X_ {L173-182} in the patient with breast cancer No. 17.

       \ vskip1.000000 \ baselineskip
    

FACS analysis of specific T-cells from Bcl-X_ {L}

The spontaneous occurrence of Bcl-X-specific CTLs L173-182 in PBL of breast cancer patients was further evaluated using FACS analysis and staining with CMH Pro5? Pentamer staining. PBL of the patient with breast cancer No. 36 with peptide was stimulated once in vitro and CD8 positive cells were isolated. This culture was stained with the HLA-A2 / BCL-X pentamer complex. FACS analyzes revealed an easily detectable population of pentamer-positive T cells that constituted 0.24% of the CD8 + T cells (Figure 8a). In comparison, the same CD8 + T cells showed approximately 1.4% of CD8 + T cells that secrete IFNγ, specific for Bcl-X L173-182 when analyzed by ELISPOT (Figure 8c) .

       \ vskip1.000000 \ baselineskip
    

Additional HLA-A2 restricted epitopes vs. Bcl-X (L)

PBL of cancer patients were inspected HLA-A2 + of different origin through ELISPOT versus Bcl-X_ {L118-126} (TAYQSFEQV) (SEQ ID NO: 43) (figure 9a) and Bcl-X_ {L169-178} (WMATYLNDHL) (SEQ ID NO: 46) (Figure 9b) identifying a spontaneous CTL response weak in patients with cancer of different origin compared to both peptides

       \ vskip1.000000 \ baselineskip
    

Responses restricted by HLA-A3 versus Bcl-X (L)

Additionally, the region was inspected inserted (including nine amino acids at each end) to determine HLA-A3 epitopes assumed using the HLA-A3 specific anchor residues main. Subsequently, two peptides were synthesized; Bcl-X_ {L165-173} (RIAAWMATY) (SEQ ID NO: 50) and the Bcl-X_ {L149-157} (ALCVESVDK) (SEQ ID NO 51). Next, PBL were inspected of patients with HLA-A3 + cancer of different origin by means of ELISPOT against the peptide Bcl-X_ {L165-173} (RIAAWMATY) (SEQ ID NO: 50) and the Bcl-X_ {L149-157} (ALCVESVDK) (SEQ ID NO: 51). Previously, it has been shown that this method is highly effective to identify specific CTLs of tumors in cancer patients. In fact, responses were detected of strong and frequent CTLs versus Bcl-X_ {L165-173} (RIAAWMATY) (SEQ ID NO: 50) in patients with cancer of different origin. I also know could detect a response against Bcl-X_ {L165-173} in PBL HLA-A3 + in four of the five cancer patients breast exams (patients who respond to treatment are defined as the average number of antigen specific cells ± 1/2 of the standard deviation> 25 per 10 5 cells), four of the four melanoma patients examined, two of the two patients with pancreatic cancer examined as well as one of the four patients with multiple myeloma examined (figure 10). From importantly, no response could be detected in any of the seven healthy HLA-A3 + individuals that were examined as controls (figure 10).

       \ vskip1.000000 \ baselineskip
    

Example 4 Immunogenicity of Mcl-1 in patients with Cancer Summary

In this example, it is shown that Mcl-1 is a target for cell recognition T in cancer patients. Therefore, responses from spontaneous T cells restricted by HLA-A1 and HLA-A3 against peptide epitopes derived from Mcl-1 through ELISPOT

       \ vskip1.000000 \ baselineskip
    

Introduction

Myeloid cell factor-1 (Mcl-1) is a member that inhibits the death of the Bcl-2 family that is expressed in early monocytic differentiation and can promote viability in transfection in immature myeloid cells. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization of myeloid cells. Elevated levels of Mcl-1 have been reported for several cancers in humans including prostate cancers, pancreatic cancers, melanoma, breast cancers, patients with ovarian cancer and cervical cancer, as well as chronic B-cell lymphocytic leukemia (B- CLL) and in AML and ALL after recurrence. In patients with B-CLL, higher levels of Mcl-1 are strongly correlated with not being able to achieve complete remission after treatment with a single agent. In multiple myeloma, Mcl-1 plays an important role in the survival of malignant cells. In this regard, it has been shown that mice expressing an mcl-1 transgene under the control of their own promoter develop B-cell neoplasms with high frequency, ranging from follicular lymphoma to diffuse large cell lymphoma.

       \ vskip1.000000 \ baselineskip
    

Responses restricted by HLA-A3 versus Mcl-1

To investigate if Mcl-1 is a natural target for T cells in cancer patients, the protein sequence for nona peptide epitopes and most likely HLA-A3 decameter, using the HLA-A3 specific anchor residues main. Subsequently, six deduced peptides were synthesized from Mcl-1 (Mcl-1_ {185-194} (YLREQATGAK) (SEQ ID NO: 52), Mcl-1_ {293-302} (SITDVLVRTK) (SEQ ID NO: 53), Mcl-1_ {267-276} (LISFGAFVAK) (SEQ ID NO: 54), Mcl-1_ {95-103} (RLLFFAPTR) (SEQ ID NO: 55), Mcl-1_ {300-308} (RTKRDWLVK) (SEQ ID NO: 56), Mcl-1_ {236-244} (DIKNEDDVK) (SEQ ID NO: 57)) and PBL of patients with HLA-A3 + cancer of different origin to determine the reactivity against these peptides, taking advantage of the assay ELISPOT Previously, it has been shown that this method is highly effective for the identification of specific CTLs of tumors in cancer patients. In fact, responses were detected of strong and frequent CTLs against two peptides derived from Mcl-1 in patients with cancer of different origin (Mcl-1_ {95-103} and Mcl-1_ {300-308}) (Figure 11). In total, five of the six patients with breast cancer HLA-A3 + examined hosted an answer immune against one of these two peptides of Mcl-1 Therefore, five patients with breast cancer they hosted an answer in front of Mcl-1_ {95-103} (patients who respond to treatment are defined as the average number of antigen specific cells ± 1/2 of the standard deviation > 25 per 10 5 cells), and three patients harbored one response to Mcl-1_ {300-308} (figure 11). Additionally two of the two cancer patients HLA-A3 + pancreatic examined housed a immune response against the peptide Mcl-1_ {95-103}, while one of these also reacted to Mcl-1_ {300-308}. Additionally it  examined the PBL of six patients suffering from B-CLL and a response was identified against Mcl-1_ {95-103} in two of these patients As a control, PBL of 10 healthy individuals were examined HLA-A3 +. Importantly, they were not detected responses to either the peptide Mcl-1_ {95-103} or the Mcl-1_ {300-308} in any of the healthy donors (figure 11). Similarly, they couldn't detect responses against any of the four peptides additional Mcl-1 derivatives in any of the cancer patients or healthy controls (data not shown).

       \ vskip1.000000 \ baselineskip
    

Answers restricted by HLA-A1 versus Mcl-1

To investigate if Mcl-1 is a natural target for T cells in cancer patients, the protein sequence for nona peptide epitopes and most likely HLA-A1 decamer, using the HLA-A1 specific anchor residues main. Subsequently, four peptides were synthesized Mcl-1 deductions (Mcl-1_ {166-175} (PAEEEEDDLY) (SEQ ID NO: 58), Mcl-1_ {121-129} (SPEEELDGY) (SEQ ID NO: 59), Mcl-1_ {177-185} (QSLEIISRY) (SEQ ID NO: 60), Mcl-1_ {339-347} (AGVGAGLAY) (SEQ ID NO: 61)) and PBL of patients were inspected with HLA-A1 + cancer of different origin for determine the reactivity against these peptides, taking advantage of of the ELISPOT trial. In fact, CTL responses were detected versus to two peptides derived from Mcl-1 in patients with cancer of different origin (Mcl-1_ {166-175} and Mcl-1_ {177-185}) (Figure 12). In total, three of the four patients with breast cancer HLA-A1 + examined hosted an answer immune against Mcl-1_ {177-185} and one of these also hosted an answer to Mcl-1_ {166-175} (Figure 12). Additionally one of the seven patients with melanoma hosted a immune response against the peptide Mcl-1_ {177-185}, and another one of these hosted an answer in front of Mcl-1_ {166-175}. As a control, it examined PBL of six healthy HLA-A1 + individuals. From importantly, no responses were detected against either the Mcl-1_ {166-175} peptide or the Mcl-1_ {177-185} in any of the healthy donors (figure 12).

       \ vskip1.000000 \ baselineskip
    

Responses to modified peptides

The peptide immunogenicity Mcl-1_ {300-308} restricted to HLA-A3 was increased by replacing threonine in the position 2 for a better anchoring residue of HLA-A3, specifically Leucine (Mcl-1_ {300-308} L2 (RLKRDWLVK) (SEQ ID NO: 62)). Spontaneous immune responses were detected in two patients with breast cancer versus Mcl-1_ {300-308} L2 (data not shown). Also, to generate more immunogenic epitope, it modified the peptide Mcl-1_ {177-185} restricted to HLA-A1 (QSLEIISRY) (SEQ ID NO: 60) in position 3 generating the two peptides Mcl-1_ {177-185} D3 (QSDEIISRY) (SEQ ID NO: 63) and Mcl-1_ {177-185} E3 (QSEEIISRY) (SEQ ID NO: 64).

       \ vskip1.000000 \ baselineskip
    

Discussion

Almost all malignant tumors are characterized due to defects in apoptosis signaling. This makes the malignant cells are resistant to endogenous apoptotic stimuli, as well as exogenous stimuli such as radiation and drugs Chemotherapists Defective apoptosis observed in cancers in humans often results from protein overexpression antiapoptotic in the Bcl-2 family of proteins, is say, Bcl-2, Bcl-X_ {L} and Mcl-1, Bcl-w, Bfl-1A1, Bcl-b and Bcl2-L-10. The use of such Apoptosis protein inhibitors for vaccination purposes is advantageous because the regulation by decrease or loss of expression of these proteins as some form of escape immune would alter sustained tumor growth, since tumor cell survival requires members functionally active of the Bcl-2 family. For therapeutic strategies, the target selection of antigens that play an insignificant role in relation to growth and tumor cell survival, tumor selection Antigen deficient is a well recognized limitation. Further, since the high expression of family proteins Bcl-2 in cells is correlated with the drug resistance, the combination of an immunotherapy based in the Bcl-2 family with cytotoxic chemotherapy is A very fascinating new way to treat cancer.

Proteins were explored Bcl-2, Bcl-X (L) and Mcl-1 to determine the presence of motives for peptide binding and these were used for the search for responses of specific T cells in cancer patients. To this end, it detected the reactivity of spontaneous T cells against all Bcl-2 family members in patients who suffered from unrelated tumor types, that is, cancer pancreatic, breast cancer, melanoma, AML and CLL through ELISPOT The presence of specific CD8 + cells was confirmed Bcl-X L in PBL of cancer patients using CD8 / pentamer FACS stains. Taken together, this data show that the CTL-defined epitopes of these proteins could be widely applied in therapeutic vaccination against cancer and therefore are of substantial immunotherapeutic value.

In addition, eleven of the patients with breast cancer they had specific CTLs of Bcl-2, eight of these patients were previously treated with at least one type of chemotherapy. In two patients (patients no .: 14 and 17) they could not CTL responses to the four peptides of Bcl-2 different. Both patients had received previously anti-hormonal treatment but not chemotherapy. Similarly, no one could be detected response in patients with primary localized breast cancer before of chemotherapy. Therefore, in patients with breast cancer, detected only responses against Bcl-2 in the patients who had received chemotherapy. Although the load Tumor can play an important role, this could indicate that immune responses are introduced or increased as consequence of the increase induced by the treatment of Bcl-2 expression. This points to a case in which the combination of a family-based immunotherapy Bcl-2 with cytotoxic chemotherapy could improve synergistic way the current response rates. The state of treatment of patients examined to determine responses to Bcl-X (L) and Mcl-1 was not available.

In the present study, the trial was used GrB ELISPOT to demonstrate that specific CTLs of Bcl-2 or Bcl-X (L) in PBL of patients are indeed cytotoxic effector cells. For further demonstrate this concept, T cells were enriched reactive against Bcl-2 from PBL of patients, and it was shown that the resulting T cell line could lyse T2 cells pulsed with peptides in a release assay of 51 Conventional Cr. In addition, this line of reactive T cells in front of Bcl-2 could destroy a cell line of breast cancer adjusted for HLA, while the target cells HLA-A2 negatives were not destroyed. These findings show that in fact cancer cells process and present the Bcl-2 peptide in the context of the molecule HLA-A2 Finally, these cells could be cloned isolated and were shown to react highly effectively against the peptide epitope of Bcl-2.

When peptides derived from melanocytic differentiation antigens were used for the first time to treat patients with stage IV melanoma, it was anticipated that this could lead to pronounced melanocyte destruction, which in turn would manifest clinically, ie vitiligo or retinitis. However, clinical experience showed that the incidence of vitiligo in patients who received vaccinations was not significantly higher than the incidence of melanoma-associated hypopigmentation in patients who received other forms of treatment. Additionally, no serious side effects have been reported in various vaccination campaigns against autoantigens. The data, taken together, demonstrate that cellular immune responses against the Bcl-2 family protein group are a general characteristic in cancer. In the attempt to maximize the impact of immunotherapy, a fascinating strategy would be to consider the expression profile and the significance of the prognosis of the target chosen in the particular disease, or stage of disease, that is being treated. Therefore, while the joint expression of Bcl-2, Mcl-1 and Bcl-XL is observed in some cancers, or a particular disease stage, other cancers show the exclusive expression of one or the other protein. Therefore, in some diseases such as ovarian cancer, the expression of Mcl-1, but not of Bcl-2, is associated with advanced stage and poor survival, which is why Mcl-1 could be the main antigen, while in diseases such as CLL, in which Bcl-2 and Mcl-1 are overexpressed together, the simultaneous targeting of both proteins may represent a more effective strategy than the targeting of any single molecule. Similarly, Tanaka et al described that the presence of another apoptosis-inhibiting survivin protein in breast carcinoma was strongly associated with Bcl-2 expression and a reduced apoptotic index (AI) and poor overall survival. A similar association between survivin and Bcl-2 in neuroblastoma, gastric cancer, colorectal cancer and high-grade non-Hodgkin lymphoma has been described. The potential efficacy and safety of survivin-derived peptides in therapeutic vaccination against cancer are currently being investigated in phase I / II clinical trials (J. Becker, personal communication). Therefore, a fascinating immunotherapeutic strategy would be to select both the Bcl-2 and the survivin family of proteins as a target, especially since they perform their antiapoptotic function through cell pathways.
different.

       \ vskip1.000000 \ baselineskip
    

Example 5 Peptide Vaccine

The peptides of the protein family Bcl-2 can for example be synthesized for example in the UVA Biomolecular Core installation with an NH2 end free amide terminal and a free acid terminal COOH end. Each is provided as a lyophilized peptide, which is then reconstitute in sterile water and dilute with Ringer's solution with Lactate (LR, Baxter Healthcare, Deerfield, IL) as a buffer for a final concentration of 67-80% solution Ringer with lactate in water. Then, they undergo filtration sterile these solutions are placed in glass vials of borosilicate and undergo a series of warranty studies of quality including identity confirmation, sterility, general safety and purity, according to FDA guidelines, such as defined in the IND 6453 standard. Stability tests of the peptide showed that the purity or the peptide concentration, when these solutions were stored of peptide at -20 ° C for 3 years.

In practical circumstances, patients they will receive a vaccine comprising approximately 100 µg of a HLA class I restricted peptide with or without an auxiliary peptide restricted to HLA class II. Patients get vaccinated with by example about 100 µg of class I HLA peptide in adjuvant alone, or get vaccinated with for example about 100 mug of the HLA class I restricted peptide plus 190 del of the auxiliary peptide restricted to class II. The dose is calculated upper auxiliary peptide to provide amounts equimolar of the auxiliary and cytotoxic peptides. Further, patients can get vaccinated with a longer peptide than It comprises the amino acid sequences of both peptides.

The above peptides, in 1 ml of solution aqueous, can be administered either as a solution / suspension with about 100 µg of QS-21, or as an emulsion with approximately 1 ml of Montanide adjuvant ISA-51.

Patients were immunized for example in the day 0 and months 1, 2, 3, 6, 9 and 12, with the most peptides adjuvant, for a total of seven immunizations. With rare exceptions, vaccinations are administered in the same arm with Each vaccine The peptides are preferably administered via s.c.

       \ vskip1.000000 \ baselineskip
    

Bibliography

1. Altieri , DC, Marchisio , PC, and Marchisio , C. Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest , 79: 1327-1333, 1999 .

2. Andersen , MH, L. Tan , I. Sondergaard , J. Zeuthen , T. Elliott , and JS Haurum . 2000 Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules. Tissue Antigens 55: 519.

3. Reed , JC 1998 . Bcl-2 family proteins. Oncogene 17: 3225.

4. Andersen , MH, LO Pedersen , JC Becker , and P. thor Straten . 2001 Identification of a Cytotoxic T Lymphocyte Response to the Apoptose Inhibitor Protein Survivin in Cancer Patients. Cancer Res . 61: 869.

5. Thurner , B., Roder , C., Dieckmann , D., Heuer , M., Kruse , M., Glaser , A., Keikavoussi , P., Kampgen , E., Bender , A., and Schuler , G. ( 1999 ) Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application J. Immunol. Methods 223, 1.

6. Shangary S and Johnson DE ( 2003 ) Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family. Leukemia 17: 1470-1482.

7. Rosenberg SA and Dudley ME ( 2004 ) Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. PNAS 101: 14639-14645.

8. Akatsuka Yoshiki et al. Journal of Exp. Med ., 2003 , vol. 197. No. 11.

9. Tetsuya et al. British journal of Hematology , 2004 , vol. 124. No. 5.

10. Sæterdal et al. PNAS , 2001 , vol. 98, no. 23.

11. Schmidt et al. Blood . July 15, 2003 , vol. 102. No. 2.

       \ global \ parskip0.000000 \ baselineskip
    

<110> Kæftens Bekæmpelse

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<120> Proteins that belong to the Bcl-2 family and fragments thereof and their use in cancer patients

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<130> P 985 PC00

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 64

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<170> PatentIn version 3.1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

         \ hskip1cm
      

8

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

         \ hskip1cm
      

9

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ hskip1cm
      

10

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ hskip1cm
      

eleven

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

         \ hskip1cm
      

12

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ hskip1cm
      

13

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

         \ hskip1cm
      

14

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

fifteen

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

16

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

17

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

18

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

twenty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

twenty-one

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

22

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

2. 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

24

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

25

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

26

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

28

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

29

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 24

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 24

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

31

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 25

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 25

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

32

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 26

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 26

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

33

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 27

         \ vskip0.400000 \ baselineskip
      

<211> 11

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Survivin derived peptide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 27

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

3. 4

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 28

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 28

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

35

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 29

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 29

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

36

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 30

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 30

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

37

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 31

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 31

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

38

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 32

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 32

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

39

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 33

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 33

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

40

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 34

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 34

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

41

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 35

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 35

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

42

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 36

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 36

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

43

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 37

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 37

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

44

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 38

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from ML-IAP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 38

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

Four. Five

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 39

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> HIV-1 epitope pol

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 39

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

46

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 40

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 40

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

47

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 41

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 41

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

48

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 42

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 42

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

49

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 43

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 43

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

fifty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 44

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 44

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

51

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 45

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 45

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

52

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 46

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 46

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

53

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 47

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Bcl-2 peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 47

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

54

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 48

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 48

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

55

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 49

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 49

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

56

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 50

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 50

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

57

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 51

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Bcl-XL

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 51

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

58

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 52

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 52

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

59

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 53

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 53

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

60

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 54

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 54

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

61

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 55

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 55

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

62

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 56

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 56

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

63

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 57

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 57

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

64

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 58

         \ vskip0.400000 \ baselineskip
      

<211> 10

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 58

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

65

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 59

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 59

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

66

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 60

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 60

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

67

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 61

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Peptide derived from Mcl-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 61

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

68

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 62

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Mcl-1 Peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 62

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

69

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 63

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Mcl-1 Peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 63

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

70

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 64

         \ vskip0.400000 \ baselineskip
      

<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Mcl-1 Peptide modified

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 64

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

71

Claims (39)

1. Isolated immunogenically active peptide that It comprises a maximum of 15 amino acids, derived from the protein Bcl-2, which comprises a sequence selected from the group consisting of: WLSLKTLLSL (SEQ ID NO: 6), PLFDFSWLSL (SEQ ID NO: 8), YLNRHLHTWI (SEQ ID NO: 10) and NIALWMTEYL (SEQ ID NO: 11) for your Use as medicine. 2. Peptide according to claim 1, being said peptide a class I CMH restricted peptide, which has the less one of the following characteristics;

(i)

can bind to the HLA molecule of class I to which it is restricted to an affinity, as measured by the amount of the peptide that half of the maximum recovery of the HLA class I molecule (value C 50), which is a maximum of 50 µM as determined through the joint-assembly test,

(ii)

can cause cells producing INF-? In a PBL population of a patient with cancer at a frequency of at least 1 per 10 4 PBLs such as is determined by an ELISPOT test, and / or

(iii)

it can perform the detection in situ in a tumor tissue of CTLs that are reactive with the epitope peptide.

3. Peptide according to claim 1, having a C 50 value, which is a maximum of 30 µM. 4. Peptide according to claim 2, having a C_ {50} value, which is a maximum of 20 µM. 5. Peptide according to claim 3, which has a C_ {50} value, which is a maximum of 10 µM. 6. Peptide according to any of the claims 1-5, which may cause Cellular immune response in a cancer patient. 7. Peptide according to any of the claims 1-6, which is restricted to a HLA-A molecule of CMH class I or a HLA-B of CMH class I. 8. Peptide according to claim 6, which is restricted to a HLA species of CMH class I selected from group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24. 9. Peptide according to claim 8, which is restricted to HLA-A2. 10. Peptide according to any of the claims 2 to 9, comprising a sequence selected from the WLSLKTLLSL group (SEQ ID NO: 6) and PLFDFSWLSL (SEQ ID NO: 8). 11. Peptide according to claim 7, which is restricted to a kind of HLA-B of the CMH class I selected from the group consisting of HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51. 12. Peptide according to any of the previous claims, which is a nonapeptide or a Decapeptide 13. Peptide according to any of the claims 1-12, which is derived from Bcl-2 by substitution, deletion or addition of al minus an amino acid residue. 14. Peptide according to any of the claims 5 to 13, which can cause cells producing INF-? In a PBL population of a patient with cancer at a frequency of at least 10 per 10 4 PBLs. 15. Peptide according to any of the claims 5 to 14, which can cause cells producing INF-? In a PBL population of a patient who has a cancer disease in which the protein is expressed Bcl-2 16. Peptide according to claim 15, in the that the cancer disease is selected from the group consisting of a hematopoietic malignant tumor including lymphatic leukemia chronic and chronic myeloid leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, cancer colon, pancreatic cancer and prostate cancer. 17. Vaccine composition comprising a isolated immunogenically active peptide derived from Bcl-2 according to claim 1-16, or a nucleic acid encoding said peptide, wherein said peptide is a class I CMH restricted peptide that has the less one of the following characteristics;

(i)

can bind to the HLA molecule of class I to which it is restricted to an affinity as measured by the amount of the peptide that half of the maximum recovery of the HLA class I molecule (value C 50) which is a maximum of 50 µM as determined by the union-assembly test as described in this document,

(ii)

can cause cells producing INF-? In a PBL population of a patient with cancer at a frequency of at least 1 per 10 4 PBL such as is determined by an ELISPOT test, and / or

(iii)

can perform in situ detection on a tumor tissue of CTL that are reactive with the epitope peptide,

For use as a medicine. 18. Vaccine composition according to claim 17, wherein the vaccine causes production in a patient vaccinated with effector T cells that have an effect cytotoxic against cancer cells. 19. Vaccine composition according to any of claims 17-18, wherein the composition It comprises an adjuvant. 20. Vaccine composition according to any of claims 17-19, comprising a peptide which is restricted to an HLA-A molecule of the CMH of class I in combination with a peptide that is restricted to a HLA-B molecule of CMH class I. 21. Vaccine composition according to any of claims 17-20, further comprising a immunogenic peptide or protein selected from survivin, ML-IAP or TRAG-3 or derived peptides thereof. 22. Vaccine composition according to any of claims 19-21, wherein the adjuvant is selected from the group consisting of DNA-based adjuvants bacterial, oil-based adjuvants / surfactant, adjuvants to base of double stranded viral RNA and imidazoquinolines. 23. Parts kit comprising the composition of vaccine according to any of claims 17 to 22 and a additional antineoplastic agent. 24. Parts kit according to claim 23, in which the antineoplastic agent is an antibody. 25. Kit of parts according to claim 23, in the one that the antineoplastic agent is a cytokine. 26. Use of the peptide according to any of claims 1-16, in a composition for ex vivo or in situ diagnosis of the presence in a patient with T-cell cancer in PBL or in tumor tissue that are reactive with Bcl-2. 27. Use of the peptide according to any of the claims 1-16, or the vaccine composition according to any of claims 17-22, in the manufacture of a medicine for treatment or prevention of a cancer disease. 28. Use according to 27, in which the disease that goes to be treated is a cancer disease in which a member of the Bcl-2 family of proteins. 29. Use according to any of the claims 28, in which the cancer disease is selected from the group that It consists of a hematopoietic malignant tumor including leukemia chronic lymphatic and chronic myeloid leukemia, melanoma, cancer breast, cervical cancer, ovarian cancer, lung cancer, cancer of colon, pancreatic cancer and prostate cancer. 30. Use according to any of the claims 27-29, which is combined with a treatment against additional cancer 31. Use according to 30, in which the treatment additional is selected from the group consisting of chemotherapy, radiotherapy, treatment with immunostimulant substances, therapy gene, antibody treatment and treatment using cells dendritic 32. Peptide according to any of the claims 1-16 or vaccine composition according to any of claims 17-22, for its use as a medicine for the treatment or prevention of a cancer disease 33. Medication according to claim 32, in which cancer disease is selected from the group consisting in a hematopoietic malignant tumor including lymphatic leukemia chronic and chronic myeloid leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, cancer colon, pancreatic cancer and prostate cancer. 34. Peptide according to any of claims 1-16, for use in a method for ex vivo or in situ diagnosis of the presence in a patient with T-cell cancer in PBL or in tumor tissue that are reactive with Bcl-2 . 35. Peptide according to any of the claims 1-16 or vaccine composition according to any of claims 17-22, for its use in a method for the treatment or prevention of a cancer disease 36. Peptides or vaccine composition according to claim 35, the cancer disease being a cancer in the that a member of the protein family is expressed Bcl-2 37. Peptides or vaccine composition according to claim 35, wherein the cancer disease is selected from the group consisting of a hematopoietic malignant tumor, including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervical cancer, ovarian cancer, cancer of lung, colon cancer, pancreatic cancer and cancer of prostate. 38. Peptides or vaccine composition according to claim 35, combining in the treatment with a treatment against additional cancer. 39. Peptides or vaccine composition according to claim 38, wherein the additional treatment is selected from the group consisting of chemotherapy, radiotherapy, treatment with immunostimulant substances, gene therapy, treatment with antibodies and treatment using dendritic cells.